BMP-7 peptides and methods of use

ABSTRACT

The invention relates to truncated BMP-7 growth factors and variants thereof. The invention also relates to methods of making and using the truncated BMP-7 growth factors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of PCT/US2011/029794,filed Mar. 24, 2011, which claims the benefit of U.S. ProvisionalApplication 61/317,072 filed Mar. 24, 2010 which is hereby incorporatedby reference in its entirety.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled“067949-5040_SequenceListing.txt,” created on or about 21 Sep. 2012,with a file size of about 11 kb contains the sequence listing for thisapplication and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The invention relates to truncated growth factors and variants thereof.The invention also relates to methods of making and using the truncatedgrowth factors.

SUMMARY OF THE INVENTION

The invention relates to isolated peptides of 138 residues or less, withthe peptide comprising an amino acid sequence at least 95% identical tothe amino acid sequence of SEQ ID NO: 2 or 3.

The invention also relates to fusion proteins comprising the inventivepeptides described herein fused to a second peptide, wherein the secondpeptide comprises an amino acid sequence that is less than 70% identicalto the amino acid sequence of SEQ ID NO: 6.

The invention also relates to methods of promoting osteoinductivity orchondroinductivity, with the methods comprising contacting cells withthe inventive peptides described herein.

The invention also relates to methods of administering the inventivepeptides described herein to matrices.

The invention also relates to methods of increasing osteogenesis orchondrogenesis in cells comprising administering to the cells theinventive peptides described herein.

The invention also relates to methods of increasing a cellular growthfactor activity comprising administering to the cells at least oneprotease and the growth factor comprising SEQ ID NO: 2, 3, 4, or 5. Insome embodiments, the composition comprises two or more proteases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts myoblast expression of alkaline phosphatase after beingcultured with modified rhBMP-7. Myoblasts treated with modified rhBMP-7showed significantly greater alkaline phosphatase activity overmyoblasts treated with unmodified rhBMP-7. When fractionated via HPLC,fractions 43 induced the highest levels of alkaline phosphatase inmyoblasts.

FIG. 2 depicts expression of alkaline phosphatase in myoblasts afterbeing cultured with various concentrations or modified rhBMP-7.

FIG. 3 depicts expression of alkaline phosphatase in myoblasts afterbeing cultured with unmodified rhBMP-7 or trypsin-modified rhBMP-7.

FIG. 4 depicts in vitro alkaline phosphatase expressions by myoblastcells cultured with non-treated rhBMP-7 control, dispase modifiedrhBMP-7, or dispase modified rhBMP-7 with the addition of collagenase inculture media.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to isolated polypeptides. In some embodiments, theisolated polypeptides are 138 amino acid residues or less. The terms“peptide,” “polypeptide” and “protein” are used interchangeably herein.As used herein, an “isolated polypeptide” is intended to mean apolypeptide that has been completely or partially removed from itsnative environment. For example, polypeptides that have been removed orpurified from cells are considered isolated. In addition, recombinantlyproduced polypeptides molecules contained in host cells are consideredisolated for the purposes of the present invention. Moreover, a peptidethat is found in a cell, tissue or matrix in which it is not normallyexpressed or found is also considered as “isolated” for the purposes ofthe present invention. Similarly, polypeptides that have beensynthesized are considered to be isolated polypeptides. “Purified,” onthe other hand is well understood in the art and generally means thatthe peptides are substantially free of cellular material, cellularcomponents, chemical precursors or other chemicals beyond, perhaps,buffer or solvent. “Substantially free” is not intended to mean thatother components beyond the novel peptides are undetectable. Thepeptides of the present invention may be isolated or purified.

The amino acid sequence of SEQ ID NO: 1 represents the full length“prepropeptide” of bone morphogenic protein 7 (“BMP-7”). Like mostmembers of the BMP family of peptides, BMP-7 is formed as a full lengthprepropeptide, which usually contains, from N-terminus to C-terminus, asignal sequence, a propeptide domain and the “mature” peptide. For BMP-7the signal sequence occurs from amino acid residues 1-29, the propeptidedomain is from residues 30 to 292, and the “mature” peptide is fromresidue 293-431 of SEQ ID NO: 1. The amino acid sequence of hBMP-7 isalso available within the UniProt Consortium Database as UniProtAccession No. P18075 the entire record of which is incorporated byreference. Normally, BMP-7, like other members of the BMP family ofgrowth factors is translated into the full length prepropeptide. Throughsubsequent post-translational processing, the signal peptide and thepropeptide domains are cleaved with the remaining portion of the peptiderecognized as the “mature” form of the growth factor. After processing,the “mature” form of the protein normally dimerizes and activehomodimeric or even heterodimeric bone morphogenetic proteins aresecreted from cells and this dimer, in general, binds to its receptor,e.g., BMP receptor type II (BMPR2), to initiate the cell signalingcascade typically associated with BMP-7 activity.

The amino acid sequences of SEQ ID NO: 2 and 3 represent noveltruncations of the mature BMP-7 and corresponds to amino acid residues328-431 of SEQ ID NO: 1 and 320-431 of SEQ ID NO: 1, respectively.Alternatively, the truncated BMP-7 can have an amino acid sequence ofSEQ ID NO: 4 or 5. As used herein, “modified growth factor” or“truncated growth factor” refers to these novel truncations of any formof BMP-7, including the full length prepropeptide, the propeptide (thefull length peptide without the signaling sequence) and the mature formof BMP-7. The invention therefore provides isolated peptides of 138residues or less, with the peptide comprising an amino acid sequence atleast about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 2 or 3. In one specific embodiment, the isolated peptide is138 residues or less and comprises an amino acid sequence at least 95%identical to the amino acid sequence of SEQ ID NO: 2 or 3. In furtherembodiments, the peptides of the present invention are 138 residues orless and comprises an amino acid sequence 100% identical to the aminoacid sequence of SEQ ID NO: 2 or 3.

In still further embodiments, the isolated peptide is 138, 137, 136,135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122,121, 120, 119, 118, 117, 116, 115, 114, 113 or 112 residues or less witheach peptide independently comprising an amino acid sequence at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2 or 3. In even more embodiments, the peptide variants described hereinstill retain their ability to specifically interact, at least partially,with its corresponding bone morphogenic protein receptor (BMPR). Inadditional embodiments, the peptide variants described herein arefunctional and capable of promoting osteoinductivity, stimulatingproliferation of osteoblasts and/or promoting osteogenesis. In even moreembodiments, the modified growth factors of the present invention haveenhanced activity compared to the unmodified growth factors. In stilleven more embodiments, the peptides consist of the amino acid sequenceof SEQ ID NO: 2 or 3, i.e., 104 amino acids in length and 100% identicalto SEQ ID NO:2, and 112 amino acids in length and 100% identical to SEQID NO: 3.

A polypeptide having an amino acid sequence at least, for example, about95% “identical” to a reference an amino acid sequence, e.g., SEQ ID NO:2 or 3, is understood to mean that the amino acid sequence of thepolypeptide is identical to the reference sequence except that the aminoacid sequence max include up to about five modifications per each 100amino acids of the reference amino acid sequence. In other words, toobtain a peptide having an amino acid sequence at least about 95%identical to a reference amino acid sequence, up to about 5% of theamino acid residues of the reference sequence may be deleted orsubstituted with another amino acid or a number of amino acids up toabout 5% of the total amino acids in the reference sequence may beinserted into the reference sequence. These modifications of thereference sequence may occur at the N-terminus or C-terminus positionsof the reference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among amino acids in thereference sequence or in one or more contiguous groups within thereference sequence.

As used herein, “identity” is a measure of the identity of nucleotidesequences or amino acid sequences compared to a reference nucleotide oramino acid sequence. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using well known techniques. While thereare several methods to measure identity between two polynucleotide orpolypeptide sequences, the term “identity” is well known to skilledartisans (Carillo (1988) J. Applied Math. 48, 1073). Examples ofcomputer program methods to determine identity and similarity betweentwo sequences include, but are not limited to, GCG program package(Devereux (1984) Nucleic Acids Research 12, 387), BLASTP, ExPASy,BLASTN, FASTA (Atschul (1990) J. Mol. Biol. 215, 403) and FASTDB.Examples of methods to determine identity and similarity are discussedin Michaels (2011) Current Protocols in Protein Science, Vol. 1, JohnWiley & Sons.

In one embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is BLASTP. Inanother embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is FASTDB, which isbased upon the algorithm of Brutlag (1990) Comp. App. Biosci. 6,237-245). In a FASTDB sequence alignment, the query and referencesequences are amino sequences. The result of sequence alignment is inpercent identity. In one embodiment, parameters that may be used in aFASTDB alignment of amino acid sequences to calculate percent identityinclude, but are not limited to: Matrix=PAM, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject amino sequence, whichever is shorter.

If the reference sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, but notbecause of internal additions or deletions, a manual correction can bemade, because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the reference sequence whencalculating percent identify. For query sequences truncated at the N- orC-termini, relative to the reference sequence, the percent identity iscorrected by calculating the number of residues of the query sequencethat are N- and C-terminus to the reference sequence that are notmatched/aligned, as a percent of the total bases of the query sequence.The results of the FASTDB sequence alignment determinematching/alignment. The alignment percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score can be used for the purposes of determining howalignments “correspond” to each other, as well as percentage identity.Residues of the reference sequence that extend past the N- or C-terminiof the query sequence may be considered for the purposes of manuallyadjusting the percent identity score. That is, residues that are notmatched/aligned with the N- or C-termini of the comparison sequence maybe counted when manually adjusting the percent identity score oralignment numbering.

For example, a 90 amino acid residue query sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the query sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe reference sequence (number of residues at the N- and C-termini notmatched/total number of residues in the reference sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched (100%alignment) the final percent identity would be 90% (100% alignment-10%unmatched overhang). In another example, a 90 residue query sequence iscompared with a 100 reference sequence, except that the deletions areinternal deletions. In this case the percent identity calculated byFASTDB is not manually corrected, since there are no residues at the N-or C-termini of the subject sequence that are not matched/aligned withthe query. In still another example, a 110 amino acid query sequence isaligned with a 100 residue reference sequence to determine percentidentity. The addition in the query occurs at the N-terminus of thequery sequence and therefore, the FASTDB alignment may not show amatch/alignment of the first 10 residues at the N-terminus. If theremaining 100 amino acid residues of the query sequence have 95%identity to the entire length of the reference sequence, the N-terminaladdition of the query would be ignored and the percent identity of thequery to the reference sequence would be 95%.

As used herein, the terms “correspond(s) to” and “corresponding to,” asthey relate to sequence alignment, are intended to mean enumeratedpositions within the reference protein, e.g., wild-type BMP-7, and thosepositions in the modified BMP-7 that align with the positions on thereference protein. Thus, when the amino acid sequence of a subject BMP-7is aliened with the amino acid sequence of a reference BMP-7, e.g., SEQID NO: 2 or 3, the amino acids in the subject sequence that “correspondto” certain enumerated positions of the reference sequence are thosethat align with these positions of the reference sequence e.g., SEQ IDNO: 2 or 3 but are not necessarily in these exact numerical positions orthe reference sequence. Methods for aligning sequences for determiningcorresponding amino acids between sequences are described herein.Accordingly, the invention provides novel peptides whose sequencescorrespond to the sequence of SEQ ID NO: 2 or 3.

The invention further embraces other species, preferably mammalian,homologs with amino acid sequences that correspond to the modifiedgrowth factors of the present invention. Species homologs, sometimesreferred to as “orthologs,” in general, share at least 35%, 40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity with the human version of the growth factors. Suchcorresponding sequences account for the modified growth factor fromacross a variety of species, such as canine, feline, mouse, rat, rabbit,monkey, etc. of BMP-7. In another embodiment, the invention providesnovel peptides whose sequences correspond to the sequence of SEQ ID NO:2 or 3 and retain at least some minimal function.

Modified growth factor products with an additional methionine residue atposition-1 (Met⁻¹-peptide) are contemplated, as are variants withadditional methionine and lysine residues at positions −2 and −1(Met⁻²-Lys⁻¹-peptide). Variants of the modified growth factor withadditional Met, Met-Lys, or Lys residues (or one or more basic residuesin general) are particularly useful for enhanced recombinant proteinproduction in bacterial host cells.

Variants resulting from insertion of the polynucleotide encoding themodified growth factor into an expression vector system are alsocontemplated. For example, variants (usually insertions) may arise fromwhen the amino terminus and/or the carboxy terminus of modified growthfactor is/are fused to another polypeptide.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in the modified growth factor peptide areremoved. Deletions can be effected at one or both termini of themodified growth factor peptide, or with removal of one or morenon-terminal amino acid residues of the modified growth factor peptide.Deletion variants, therefore, include all fragments of the modifiedgrowth factor peptide.

Within the confines of the disclosed percent identity, the inventionalso relates to substitution variants of disclosed polypeptides of theinvention. Substitution variants include those polypeptides wherein oneor more amino acid residues of truncated growth factor are removed andreplaced with alternative residues. In one aspect, the substitutions areconservative in nature; however, the invention embraces substitutionsthat are also non-conservative. Conservative substitutions for thispurpose may be defined as set out in the tables below. Amino acids canbe classified according to physical properties and contribution tosecondary and tertiary protein structure. A conservative substitution isrecognized in the art as a substitution of one amino acid for anotheramino acid that has similar properties. Exemplary conservativesubstitutions are set out in below.

TABLE I Conservative Substitutions Side Chain Characteristic Amino AcidAliphatic Non-polar Gly, Ala, Pro, Iso, Leu, Val Polar-uncharged Cys,Ser, Thr, Met, Asn, Gln Polar-charged Asp, Glu, Lys, Arg Aromatic His,Phe, Trp, Tyr Other Asn, Gln, Asp, Glu

Alternatively, conservative amino acids can be grouped as described inLehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp.71-77, as set forth below.

TABLE II Conservative Substitutions Side Chain Characteristic Amino AcidNon-polar (hydrophobic) Aliphatic: Ala, Leu, Iso, Val, Pro Aromatic:Phe, Trp Sulfur-containing: Met Borderline: Gly Uncharged-polarHydroxyl: Ser, Thr, Tyr Amides: Asn, Gln Sulfhydryl: Cys Borderline: GlyPositively Charged (Basic): Lys, Arg, His Negatively Charged (Acidic)Asp, Glu

And still other alternative, exemplary conservative substitutions areset out below.

TABLE III Conservative Substitutions Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

The polypeptides of the present invention may or may not be involved ina dimer. In one embodiment, the invention provides dimers, wherein thedimers comprise at least one of the novel, modified growth factorsdescribed herein. In one specific embodiment, the dimers are homodimersof the novel modified growth factors. In another embodiment, the dimersare heterodimers comprising at least one of the novel modified growthfactors. As used herein, a “heterodimer” means a dimer of two peptides,wherein the amino acid sequences of the peptides are not 100% identicalto each other. Thus, a heterodimer may include a modified growth factorpeptide of the present invention dimerized with a normal, “mature”version of the same growth factor.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. In some embodiments, at least one refers, for example, to1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

It should be understood that the definition of peptides or polypeptidesof the invention is intended to include polypeptides bearingmodifications other than insertion, deletion, or substitution of aminoacid residues. By way of example, the modifications may be covalent innature, and include for example, chemical bonding with polymers, lipids,other organic and inorganic moieties. Such derivatives may be preparedto increase circulating half-life of a polypeptide, or may be designedto improve the targeting capacity of the polypeptide for desired cells,tissues or organs. Similarly, the invention further embraces modifiedgrowth factor peptides that have been covalently modified to include oneor more water-soluble polymer attachments such as polyethylene glycol,polyoxyethylene glycol or polypropylene glycol.

Chemically modified growth factor compositions in which the modifiedgrowth factor is linked to a polymer are included within the scope ofthe present invention. The polymer may be water soluble to preventprecipitation of the protein in an aqueous environment, such as aphysiological environment. Suitable water-soluble polymers may beselected from the group consisting of, for example, polyethylene glycol(PEG), monomethoxypolyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, polypropylene glycol homopolymers, a polypropyleneoxide/ethylene oxide copolymer polyoxyethylated polyols (e.g., glycerol)and polyvinyl alcohol. The selected polymer is usually modified to havea single reactive group, such as an active ester for acylation or analdehyde for alkylation, so that the degree of polymerization may becontrolled. Polymers may be of any molecular weight and may be branchedor unbranched, and mixtures of such polymers may also be used. When thechemically modified NgR polymer is destined for therapeutic use,pharmaceutically acceptable polymers will be selected for use.

Pegylation of modified growth factor peptides may be carried out by anyof the pegylation reactions known in the art. Preferably, the pegylationis carried out via an acylation reaction or an alkylation reaction witha reactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer). A preferred water-soluble polymer for pegylationof polypeptides is polyethylene glycol (PEG), including, but not limitedto bi-functional PEGs. As used herein, “polyethylene glycol” is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (Cl-ClO) alkoxy- or aryloxy-polyethyleneglycol.

Chemical derivatization of modified growth factor may be performed underany suitable conditions used to react with a biologically activesubstance with an activated polymer molecule. Methods for preparingpegylated modified growth factor will generally comprise the steps of(a) reacting the polypeptide with polyethylene glycol, such as areactive ester or aldehyde derivative of PEG, under conditions wherebymodified growth factor polypeptide becomes attached to one or more PEGgroups, and (b) obtaining the reaction products. It will be apparent toone of ordinary skill in the art to select the optimal reactionconditions or the acylation reactions based on known parameters and thedesired result.

Pegylated and other polymer modified growth factor polypeptides maygenerally be used to treat conditions that may be alleviated ormodulated by administration of the modified growth factor polypeptidesdescribed herein. However, the chemically-derivatized polymer: modifiedgrowth factor polypeptide molecules disclosed herein may have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the nonderivatized molecules. The modified growth factorpolypeptides, fragments thereof, variants and derivatives, may beemployed alone, together, or in combination with other pharmaceuticalcompositions. For example, cytokines, growth factors, antibiotics,anti-inflammatories and/or chemotherapeutic agents may beco-administered as is appropriate for the indication being treated.

The present invention provides compositions comprising purifiedpolypeptides of the invention. Preferred compositions comprise, inaddition to the polypeptide of the invention, a pharmaceuticallyacceptable (i.e. sterile and non-toxic) liquid, semisolid, or soliddiluent that serves as a pharmaceutical vehicle, excipient or medium.Any diluent known in the art may be used. Exemplary diluents include,but are not limited to, water, saline solutions, polyoxyethylenesorbitan monolaurate, magnesium stearate, methyl- and propyhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose,sorbitol, mannitol, glycerol, calcium phosphate, mineral oil and cocoabutter.

In one embodiment, the invention provides fusion proteins comprising atleast a first and a second fusion peptide. The fusion partners are,generally speaking, covalently bonded to one another via a typical aminebond between the fusion peptides, thus creating one contiguous aminoacid chain. In one specific embodiment, the first peptide of the fusionprotein comprises a peptide of 138 residues or less, with the peptidecomprising an amino acid sequence at least about 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 2 or 3. In onespecific embodiment, the first fusion peptide is 138 residues or lessand comprises an amino acid sequence at least 95% identical to the aminoacid sequence of SEQ ID NO: 2 or 3. In further embodiments, the firstfusion peptide is 138 residues or less and comprises an amino acidsequence 100% identical to the amino acid sequence of SEQ ID NO: 2 or 3.In still further embodiments, the first fusion peptide is 138, 137, 136,135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122,121, 120, 119, 118, 117, 116, 115, 114, 113 or 112 with each firstfusion peptide independently comprising an amino acid sequence at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%,97%, 98%, 990% or 100% identical to the amino acid sequence of SEQ IDNO: 3. In even more embodiments, the first fusion peptide consist of theamino acid sequence of SEQ ID NO: 3, i.e., 112 amino acids in length and100% identical to SEQ ID NO: 3. In still further embodiments, the firstfusion peptide is 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128,127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114,113, 112, 111, 110, 109, 108, 107, 106, 105 or 104 residues or less witheach first fusion peptide independently comprising an amino acidsequence at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 2. In even more embodiments, the first fusionpeptide consist of the amino acid sequence of SEQ ID NO: 2, i.e., 104amino acids in length and 100% identical to SEQ ID NO: 2.

In one specific embodiment, the second fusion peptide comprises an aminoacid sequence that does not include the amino acid sequence of SEQ IDNO: 6. In more specific embodiments, the second fusion of the presentinvention comprises an amino acid sequence that is less than 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% identical to SEQ ID NO: 6.

In another specific embodiment, the second fusion peptide comprises anamino acid sequence that does not include the amino acid sequence of SEQID NO: 7. In more specific embodiments, the second fusion of the presentinvention comprises an amino acid sequence that is less than 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% identical to SEQ ID NO: 7.

Other types of fusion proteins provided by the present invention includebut are not limited to, fusions with secretion signals and otherheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the protein to improve stability and persistence inthe host cell, during purification or during subsequent handing andstorage.

Additional fusion proteins include fusions for enhancing translocationof the protein across cell membranes. For example, Tat is an 86-aminoacid protein involved in the replication of human immunodeficiency virustype (HIV-1). The HIV-1 Tat transactivation protein is efficiently takenup by cells, and it has been demonstrated that low concentrations (nM)are sufficient to transactivate a reporter gene expressed from the HIV-1promoter. Exogenous Tat protein is able to translocate through theplasma membrane and reach the nucleus to transactivate the viral genome.Tat peptide-mediated cellular uptake and nuclear translocation have beendemonstrated in several systems. Chemically coupling a Tat-derivedpeptide (residues 37-72 of Tat) to several proteins results in theirinternalization in several cell lines or tissues (Fawell (1994) Proc.Natl. Acad. Sci. USA 91, 664-668.

It is well-known that a region of the Tat protein centered on a clusterof basic amino acids is responsible for this translocation activity. Asynthetic peptide consisting of the Tat basic amino acids 48-60 with acysteine residue at the C-terminus coupled to fluorescein maleimidetranslocates to the cell nucleus as determined by fluorescencemicroscopy. In addition, a fusion protein (Tat-NLS-β-Gal) consisting ofTat amino acids 48-59 fused by their amino-terminus to β-galactosidaseamino acids 9-1023 translocates to the cell nucleus in an ATP-dependent,cytosolic factor-independent manner. Accordingly, the fusion proteins ofthe present invention may comprise all or a portion of HIV-Tat, such asany sequential residues of the Tat protein basic peptide motif 37-72(37-CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72 (SEQ ID NO: 8). The minimumnumber of amino acid residues can be in the range of from about three toabout six. In one embodiment, the Tat portion of the fusion protein isfrom about three to about five contiguous amino acids in length. Inanother embodiment, the Tat portion of the fusion protein is about fouramino acids in length, i.e., the minimal requirement for one alphahelical turn. In another embodiment, the Tat portion of the fusionprotein comprises Tat protein residues 48-57 (GRKKRRQRRR) (SEQ ID NO:9).

In additional embodiments of fusion proteins, a region may be added tofacilitate purification. For example, “histidine tags” (“his tags”) or“lysine tags” (the second fusion peptide) may be appended to the firstfusion peptide. Examples of histidine tags include, but are not limitedto hexaH, heptaH and hexaHN. Examples of lysine tags include, but arenot limited to pentaL, heptaL and FLAG. Such regions may be removedprior to final preparation of the protein. Other examples of a secondfusion peptide include, but are not limited to glutathione S-transferase(GST) and alkaline phosphatase (AP).

The addition of peptide moieties to proteins, whether to engendersecretion or excretion, to improve stability and to facilitatepurification or translocation, among others, is a familiar and routinetechnique in the art and may include modifying amino acids at theterminus to accommodate the tags. For example in SEQ ID NO: 2 or 3, theN-terminus amino acid may be modified to, for example, arginine and/orserine to accommodate a tag. Of course, the amino acid residues of theC-terminus may also be modified to accommodate tags. One particularlyuseful fusion protein comprises a heterologous region fromimmunoglobulin that can be used solubilize proteins. For example, EPA0464 533 discloses fusion proteins comprising various portions ofconstant region of immunoglobin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and therebyresults, for example, in improved pharmacokinetic properties (EP A0232262). On the other hand, for some uses, it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described.

The fusion proteins of the current invention can be recovered andpurified from recombinant cell cultures by well-known methods including,but not limited to, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, e.g., immobilized metal affinity chromatography (IMAC),hydroxylapatite chromatography and lectin chromatography. Highperformance liquid chromatography (“HPLC”) may also be employed forpurification. Well-known techniques for refolding protein may beemployed to regenerate active conformation when the fusion protein isdenatured during isolation and/or purification.

Fusion proteins of the present invention include, but are not limitedto, products of chemical synthetic procedures and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the fusion proteins of the present invention may be glycosylated or maybe non-glycosylated. In addition, fusion proteins of the invention mayalso include an initial modified methionine residue, in some cases as aresult of host-mediated processes.

The invention also relates to isolated nucleic acids and to constructscomprising these nucleic acids. The nucleic acids of the invention canbe DNA or RNA, for example, mRNA. The nucleic acid molecules can bedouble-stranded or single-stranded; single stranded RNA or DNA can bethe coding, or sense, strand or the non-coding, or antisense, strand. Inparticular, the nucleic acids may encode any polypeptide of theinvention, including, but not limited to, the fusion proteins of thepresent invention. For example, the nucleic acids of the inventioninclude polynucleotide sequences that encode glutathione-S-transferase(GST) fusion protein, poly-histidine (e.g. His₆), poly-HN poly-lysine,hemagglutinin, HSV-Tag and at least a portion of HIV-Tat. If desired,the nucleotide sequence of the isolated nucleic acid can includeadditional non-coding sequences such as non-coding 3′ and 5′ sequences(including regulatory sequences, for example).

In some embodiments, the isolated nucleic acid of the present inventionfurther relates to the nucleic acids consisting of a nucleic acidsequence at least 95% identical to the nucleic acid sequence of SEQ IDNO: 10 or 11. In further embodiments, the invention therefore providesthe nucleic acids consisting of a nucleic acid sequence at least about75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ IDNO: 10 or 11.

The nucleic acid molecules of the invention can be “isolated.” As usedherein, an “isolated” nucleic acid molecule or nucleotide sequence isintended to mean a nucleic acid molecule or nucleotide sequence that isnot flanked by nucleotide sequences normally flanking the gene ornucleotide sequence (as in genomic sequences) and/or has been completelyor partially removed from its native environment (e.g. a cell, tissue).For example, nucleic acid molecules that have been removed or purifiedfrom cells are considered isolated. In some instances, the isolatedmaterial will form part of a composition (for example, a crude extractcontaining other substances), buffer system or reagent mix. In othercircumstances, the material may be purified to near homogeneity, forexample as determined by PAGE or column chromatography such as HPLC.Thus, an isolated nucleic acid molecule or nucleotide sequence canincludes a nucleic acid molecule or nucleotide sequence which issynthesized chemically, using recombinant DNA technology or using anyother suitable method. To be clear, a nucleic acid contained in a vectorwould be included in the definition of “isolated” as used herein. Also,isolated nucleotide sequences include recombinant nucleic acid molecules(e.g. DNA, RNA) in heterologous organisms, as well as partially orsubstantially purified nucleic acids in solution. “Purified,” on theother hand is well understood in the art and generally means that thenucleic acid molecules are substantially free of cellular material,cellular components, chemical precursors or other chemicals beyond,perhaps, buffer or solvent. “Substantially free” is not intended to meanthat other components beyond the novel nucleic acid molecules areundetectable. The nucleic acid molecules of the present invention may beisolated or purified. Both in vivo and in vitro RNA transcripts of a DNAmolecule of the present invention are also encompassed by “isolated”nucleotide sequences.

The invention also encompasses variations of the nucleotide sequences ofthe invention, such as those encoding functional fragments or variantsof the polypeptides as described above. Such variants can benaturally-occurring, or non-naturally-occurring, such as those inducedby various mutagens and mutagenic processes. Intended variationsinclude, but are not limited to, addition, deletion and substitution ofone or more nucleotides which can result in conservative ornon-conservative amino acid changes, including additions and deletions.

The invention described herein also relates to fragments of the isolatednucleic acid molecules described herein. The term “fragment” is intendedto encompass a portion of a nucleotide sequence described herein whichis from at least about 20 contiguous nucleotides to at least about 50contiguous nucleotides or longer in length. Such fragments may be usefulas probes and primers. In particular primers and probes may selectivelyhybridize to the nucleic acid molecule encoding the polypeptidesdescribed herein. For example, fragments which encode polypeptides thatretain activity, as described below, are particularly useful.

The invention also provides nucleic acid molecules that hybridize underhigh stringency hybridization conditions, such as for selectivehybridization, to the nucleotide sequences described herein (e.g.,nucleic acid molecules which specifically hybridize to a nucleotidesequence encoding polypeptides described herein and encode a modifiedgrowth factor). Hybridization probes include synthetic oligonucleotideswhich bind in a base-specific manner to a complementary strand ofnucleic acid.

Such nucleic acid molecules can be detected and/or isolated by specifichybridization e.g. under high stringency conditions. “Stringencyconditions” for hybridization is a term of art that refers to theincubation and wash conditions, e.g. conditions of temperature andbuffer concentration, which permit hybridization of a particular nucleicacid to a second nucleic acid; the first nucleic acid may be perfectlycomplementary, i.e., 100%, to the second, or the first and second mayshare some degree of complementarity, which is less than perfect, e.g.,60%, 75%, 85%, 95% or more. For example, certain high stringencyconditions can be used which distinguish perfectly complementary nucleicacids from those of less complementarity.

“High stringency conditions”, “moderate stringency conditions” and “lowstringency conditions” for nucleic acid hybridizations are explained inCurrent Protocols in Molecular Biology, John Wiley & Sons). The exactconditions which determine the stringency of hybridization depend notonly on ionic strength, e.g., 0.2×SSC, 0.1×SSC of the wash buffers,temperature, e.g., room temperature, 42° C. 68° C., etc., and theconcentration of destabilizing agents such as formamide or denaturingagents such as SDS, but also on factors such as the length of thenucleic acid sequence, base composition, percent mismatch betweenhybridizing sequences and the frequency of occurrence of subsets of thatsequence within other non-identical sequences. Thus, high, moderate orlow stringency conditions mat be determined empirically.

By varying hybridization conditions from a level of stringency at whichno hybridization occurs to a level at which hybridization is firstobserved, conditions which will allow a given sequence to hybridize withthe most similar sequences in the sample can be determined. Exemplaryconditions are described in Krause (1999) Methods in Enzymology200:546-556. Washing is the step in which conditions are usually set soas to determine a minimum level of complementarity of the hybrids.Generally, starting from the lowest temperature at which only homologoushybridization occurs, each degree (° C.) by which the final washtemperature is reduced, while holding SSC concentration constant, allowsan increase by 1% in the maximum extent of mismatching among thesequences that hybridize. Generally, doubling the concentration of SSCresults in an increase in Tm. Using these guidelines, the washingtemperature can be determined empirically for high, moderate or lowstringency, depending on the level of mismatch sought. Exemplary highstringency conditions include, but are not limited to, hybridization in50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60°C. Example of progressively higher stringency conditions include, afterhybridization, washing with 0.2×SSC and 0.1% SDS at about roomtemperature (low stringency conditions); washing with 0.2×SSC, and 0.1%SDS at about 42° C. (moderate stringency conditions); and washing with0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, washing may encompass two or more of the stringencyconditions in order of increasing stringency. Optimal conditions willvary, depending on the particular hybridization reaction involved, andcan be determined empirically.

Equivalent conditions can be determined by varying one or more of theparameters given as an example, as known in the art, while maintaining asimilar degree of identity or similarity between the target nucleic acidmolecule and the primer or probe used. Hybridizable nucleotide sequencesare useful as probes and primers for identification of organismscomprising a nucleic acid of the invention and/or to isolate a nucleicacid of the invention, for example. The term “primer” is used herein asit is in the art and refers to a single-stranded oligonucleotide whichacts as a point of initiation of template-directed DNA synthesis underappropriate conditions in an appropriate buffer and at a suitabletemperature. The appropriate length of a primer depends on the intendeduse of the primer, but typically ranges from about 15 to about 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the template,but must be sufficiently complementary to hybridize with a template. Theterm “primer site” refers to the area of the target DNA to which aprimer hybridizes. The term “primer pair” refers to a set of primersincluding a 5′ (upstream) primer that hybridizes with the 5′ end of theDNA sequence to be amplified and a 3′ (downstream) primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The present invention also relates to vectors that include nucleic acidmolecules of the present invention, host cells that are geneticallyengineered with vectors of the invention and the production of proteinsof the invention by recombinant techniques.

In accordance with this aspect of the invention, the vector may be, forexample, a plasmid vector, a single- or double-stranded phage vector, ora single- or double-stranded RNA or DNA viral vector. Such vectors maybe introduced into cells as polynucleotides, for example DNA, bywell-known techniques for introducing DNA and RNA into cells. Viralvectors may be replication competent or replication defective. In thelatter, case viral propagation generally will occur only incomplementing host cells.

In certain respects, the vectors to be used are those for expression ofpolynucleotides and proteins of the present invention. Generally, suchvectors comprise cis-acting control regions effective for expression ina host operatively linked to the polynucleotide to be expressed.Appropriate trans-acting factors are supplied by the host, supplied by acomplementing vector or supplied by the vector itself upon introductioninto the host.

A great variety of expression vectors can be used to express theproteins of the invention. Such vectors include chromosomal, episomaland virus-derived vectors, e.g., vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, from viruses such as adeno-associated virus,lentivirus, baculoviruses, papova viruses, such as SV40, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such asthose derived from plasmid and bacteriophage genetic elements, such ascosmids and phagemids. All may be used for expression in accordance withthis aspect of the present invention. Generally, any vector suitable tomaintain, propagate or express polynucleotides or proteins in a host maybe used for expression in this regard.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s) including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude, but are not limited to, the phage lambda PL promoter, the E.coli lac, trp and tac promoters, HIV promoters, the SV40 early and latepromoters and promoters of retroviral LTRs, to name just a few of thewell-known promoters. In general, expression constructs will containsites for transcription, initiation and termination and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the mature transcripts expressed by the constructs willinclude a translation initiating AUG at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

In addition, the constructs may contain control regions that regulate,as well as engender expression. Generally, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors may contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cells.Preferred markers include dihydrofolate reductase or neomycin resistancefor eukaryotic cell culture, and tetracycline, kanamycin or ampicillinresistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence, as well as anappropriate promoter, and other appropriate control sequences, may beintroduced into an appropriate host using a variety of well-knowntechniques suitable to expression therein of a desired polypeptide.Representative examples of appropriate hosts include bacterial cells,such as, but not limited to, E. coli, Streptomyces, Bacillus, andSalmonella cells; fungal cells, such as yeast cells; insect cells suchas Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COSand Bowes melanoma cells; and plant cells. Hosts for of a great varietyof expression constructs are well known, and those of skill in the artwill be enabled by the present disclosure to select an appropriate hostfor expressing one of the proteins of the present invention.

Examples of vectors that may be useful for fusion proteins include, butare not limited to, pGEX (Pharmacia), pMAL (New England Biolabs) andpRIT5 (Pharmacia) that fuse glutathione-S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. Often, in fusion expression vectors, a proteolytic cleavagesite is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase.

Examples of vectors for expression in yeast S. cerevisiae includepYepSec1 (Baldari (1987) EMBO J. 6, 229-234), pMFa (Kurjan (1982) Cell30, 933-943), pJRY88 (Schultz (1987) Gene 54, 115-123), pYES2(Invitrogen) and picZ (Invitrogen).

Alternatively, the modified growth factors can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith (1983) Mol. Cell. Biol. 3, 21562165) and the pVL series (Lucklow (1989) Virology 170, 31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329, 840)and pMT2PC (Kaufman (1987) EMBO J. 6, 187 195). When used in mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.For other suitable expression systems for both prokaryotic andeukaryotic cells, see Sambrook (2010) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include liver-specific promoters (e.g., albumin promoter),lymphoid-specific promoters such as, but not limited to, T cellreceptors and immunoglobulins, neuron-specific promoters (e.g.neurofilament promoter), pancreas-specific promoters, mammarygland-specific promoters (e.g. milk whey promoter), bone-specificpromoters (e.g., osteocalcin, osteopontin or bone sialoprotein, promoterregions), cartilage specific promoters (e.g., WARP) and muscle specificpromoters (Desmin, myglobin, etc) just to name a few.Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249, 374 379) andthe α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537546).

The present invention also relates to host cells containing theabove-described constructs. The host cell can be a higher eukaryoticcell, such as a mammalian cell, or a lower eukaryotic cell, such as ayeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. The host cell can be stably or transiently transfectedwith the construct. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention. As used herein, a “host cell” is a cell that normallydoes not contain any of the nucleotides of the present invention andcontains at least one copy of the nucleotides of the present invention.Thus, a host cell as used herein can be a cell in a culture setting orthe host cell can be in an organism setting where the host cell is partof an organism, organ or tissue.

Suitable host cells for expression of the polypeptides of the inventioninclude, but are not limited to, prokaryotes, yeast, and eukaryotes. Ifa prokaryotic expression vector is employed, then the appropriate hostcell would be any prokarytic cell capable of expressing the clonedsequences. Suitable prokaryotic cells include, but are not limited to,bacteria of the genera Escherichia, Bacillus, Pseudomonas,Staphylococcus, and Streptomyces.

If a eukaryotic expression vector is employed, then the appropriate hostcell would be any eukaryotic cell capable of expressing the clonedsequence. In one embodiment, eukaryotic cells are cells of highereukaryotes. Suitable eukaryotic cells include, but are not limited to,non-human mammalian tissue culture cells and human tissue culture cells.Other host cells include, but are not limited to, insect cells, HeLacells, Chinese hamster ovary cells (CHO cells), African green monkeykidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts.

In addition, a yeast cell may be employed as a host cell. Yeast cellsinclude, but are not limited to, the genera Saccharomyces, Pichia andKluveromyces. In one embodiment, the yeast hosts are S. cerevisiae or P.pastoris. Yeast vectors may contain an origin of replication sequencefrom a 2T yeast plasmid, an autonomously replication sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination and a selectable marker gene. Shuttle vectorsfor replication in both yeast and E. coli are also included herein.

Introduction of a construct into the host cell can be affected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Toidentify and select these integrants, a gene that encodes a selectablemarker (e.g., resistance to antibiotics) is generally introduced intothe host cells along with the gene of interest. Various selectablemarkers include those that confer resistance to drugs, such as G418,hygromycin, dihydrofolate reductase (DHFR) and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding modified growth factor or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die). In one embodiment, the polypeptides of theinvention are expressed in Chinese Hamster Ovary (CHO) cells.

The modified growth factor expression vectors prepared as above areintroduced into CHO cells by any known method, including, but notlimited to the calcium phosphate method and electroporation.

Transformants carrying the expression vectors are selected based on theabove-mentioned selectable markers. Repeated clonal selection of thetransformants using the selectable markers allows selection of stablecell lines expressing the modified growth factor constructs. Increasedconcentrations in the selection medium allows gene amplification andgreater expression of the desired modified growth factor. The hostcells, for example CHO cells, containing the recombinant modified growthfactor can be produced by cultivating the CHO cells containing themodified growth factor expression vectors constitutively expressing themodified growth factor constructs.

Accordingly, the current invention also relates to methods of producinga modified growth factor comprising culturing the host cells of theinvention under conditions such that the modified growth factor isexpressed, and recovering said protein. The culture conditions requiredto express the proteins of the current invention are dependent upon thehost cells that are harboring the polynucleotides of the currentinvention. The culture conditions for each cell type are well-known inthe art and can be easily optimized, if necessary. For example, anucleic acid encoding a polypeptide of the invention, or a constructcomprising such nucleic acid, can be introduced into a suitable hostcell by a method appropriate to the host cell selected, e.g.,transformation, transfection, electroporation, infection, such that thenucleic acid is operably linked to one or more expression controlelements as described herein. Host cells can be maintained underconditions suitable for expression in vitro or in vivo, whereby theencoded polypeptide is produced. For example host cells may bemaintained in the presence of an inducer, suitable media supplementedwith appropriate salts, growth factors, antibiotic, nutritionalsupplements, etc., which may facilitate protein expression. Inadditional embodiments, the modified growth factors of the invention canbe produced by in vitro translation of a nucleic acid that encodes themodified growth factor, by chemical synthesis or by any other suitablemethod. If desired, the modified growth factor can be isolated from thehost cell or other environment in which the protein is produced orsecreted. It should therefore be appreciated that the methods ofproducing the modified growth factors encompass expression of thepolypeptides in a host cell of a transgenic animal or plant. See U.S.Pat. Nos. 6,013,857, 5,990,385, and 5,994,616.

In situations where the modified BMP-7 will be found primarilyintracellularly, intracellular material (including inclusion bodies forGram-negative bacteria) can be extracted from the host cell using anystandard technique known to one of ordinary skill in the art. Suchmethods would encompass, by way of example and not by way of limitation,lysing the host cells to release the contents of the periplasm/cytoplasmby French press, homogenization and/or sonication followed bycentrifugation.

If the modified BMP-7 has formed inclusion bodies in the cytosol, suchinclusion bodies may frequently bind to the inner and/or outer cellularmembranes. Upon centrifugation, the inclusion bodies will be foundprimarily in the pellet material. The pellet material can then betreated at pH extremes or with one or more chaotropic agents such as adetergent, guanidine, guanidine derivatives, urea, or urea derivativesin the presence of a reducing agent such as dithiothreitol at alkalinepH or tris-carboxyethyl phosphine at acid pH to release, break apart andsolubilize the inclusion bodies. Once solubilized, the modified growthfactor peptide can be analyzed using gel electrophoresis,immunoprecipitation or the like. Various methods of isolating themodified growth factor peptide would be apparent to one of ordinaryskill in the art, for example, isolation may be accomplished usingstandard methods such as those set forth below and in Marston et al(1990) Meth. Enzymol. 182, 264-275.

If the modified growth factor peptide is not biologically activefollowing the isolation procedure employed, various methods for“refolding” or converting the polypeptide to its tertiary structure andgenerating disulfide linkages, can be used to restore biologicalactivity. Methods known to one of ordinary skill in the art includeadjusting the pH of the solubilized polypeptide to a certain pH, usuallyabove 7, and in the presence of a particular concentration of achaotrope. The selection of chaotrope is very similar to the choicesused for inclusion body solubilization, but usually at a lowerconcentration, and is not necessarily the same chaotrope as used for thesolubilization. It may be required to employ a reducing agent or thereducing agent plus its oxidized form in a specific ratio, to generate aparticular redox potential allowing for disulfide shuffling during theformation of the protein's cysteine bridge(s). Some of the commonly usedredox couples include cysteine/cysteine, glutathione (GSH)/dithiobisGSH, cupric chloride, dithiothreitol (DTT)/dithiane DTT,2-mercaptoethanol (bME)/dithio-b(ME). To increase the efficiency of therefolding, it may be necessary to employ a cosolvent, such as glycerol,polyethylene glycol of various molecular weights and arginine.

Other methods of preparing the modified growth factors of the presentinvention include, but are not limited to, contacting a form of BMP-7with a protease. In one embodiment, the methods comprise contacting themature form of BMP-7 with a protease to produce the modified growthfactors of the present invention. In one embodiment, the protease is aserine protease, a threonine protease, a metalloproteinase, a cysteineprotease, an aspartate protease or a glutamic acid protease. Examples ofsuch proteases are well known in the art and include proteases from botheukaryotic and prokaryotic sources. Examples of serine proteasesinclude, but are not limited to, chymotrypsin, trypsin, elastase,subtilisin and alpha/beta hydrolases. Examples of cysteine proteasesinclude, but are not limited to, actinidain, bromelain, calpains, somecathepsins, clostripain and papain. Examples of aspartate proteasesinclude, but are not limited to, some cathepsins, chyomsin, renin,pepsin and HIV-1 protease. Examples of metalloproteinases include, butare not limited to, the matrix metalloproteinase (MMP) family ofproteases that include MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7,MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16,MMP-17, MMP-18 and membrane-type MMP (MT-MMP), to name a few. Many ofthese MMPs are known by other names in the art and one of skill will beinformed of synonyms of the various enzymes. For example, some of the“collagenases” are MMP-1, MMP-8, MMP-13, MMP-14, MMP-18 and dispases,and some of the “gelatinases” are MMP-2, MMP-9 and MMP-12; some of the“stromelysins” are MMP-3, MMP-10 and MMP-11, and some of themembrane-type matrix metalloproteinases are MMP-14, MMP-15, MMP-16,MMP-17, MMP-24, and MMP-25. Collagenases are examples ofmetalloproteinases that break down native collagen or gelatin. A varietyof microorganisms and many different types of animal cells producecollagenases. Bacterial collagenases are often used in the laboratory todigest tissues and isolate individual cells. Collagenase produced byClostridium histolyticum (C. histolyticum) is a zinc metalloproteinasethat degrades various types of collagen and gelatin. Additional examplesof metalloproteinases are the astacin family, such as, but not limitedto, astacin, tolloid, xolloid, mammalian tolloid like (mTLL), BMP-1,meprin A and B, and matrilysin and the aggrecanase (ADAMTS) family ofmetalloproteinase, such as, but not limited to, ADAMTS-1, ADAMTS-2,ADAMTS-3, ADAMTS-4. ADAMTS-5.

Of course, the methods include contacting a form of BMP-7 with more thanone protease either sequentially or simultaneously. In addition, theproteases used herein can be recombinant or isolated from varioussources. The proteases used herein need not be isolated from the samecellular or animal source as the mature form of the BMP-7. For example,many proteases are found only in prokaryotes, yet these proteases arefunctional against proteins from eukaryotes. Factors affecting enzymeactivity include, but are not limited to, salt concentrations of buffer,pH and temperature. One of skill in the art will readily understand theconditions necessary to promote peptide cleave based upon the enzymeused.

In some embodiments, the ratio of the amount of protease(s) to theamount of growth factor(s) may be between about 1 mol protease: 1000 molgrowth factor and about 1000 mol:1 mol, between about 1 mol protease:100 mol growth factor and about 1000 mol:1 mol, between about 1 mol:10mol and about 1000 mol:1 mol, between about 1 mol: 10 mol and about 100mol:1 mol, between about 1 mol: 10 mol and about 10 mol:1 mol, orbetween about 1 mol:1 mol and about 10 mol:11 mol. In furtherembodiments, the ratio of the amount of collagenase(s) to the amount ofgrowth factor(s) may be between about 1 mol collagenase:100 mol growthfactor and about 1000 mol:1 mol, between about 1 mol:10 mol and about1000 mol:1 mol, between about 1 mol:10 mol and about 100 mol:1 mol, orbetween about 1 mol:1 mol and about 10 mol:1 mol. The ratio of theamount of trypsin to the amount of growth factor(s) may be between about1 mol trypsin: 1000 mol growth factor and about 1000 mol:1 mol, betweenabout 1 mol:100 mol and about 1000 mol:1 mol, between about 1 mol:10 moland about 100 mol:1 mol, or between about 1 mol: 10 mol and about 10mol:1 mol. The ratio of the amount of clostripain to the amount ofgrowth factor(s) may be between about 1 mol clostripain:1000 mol growthfactor and about 1000 mol:1 mol, between about 1 mol:100 mol and about1000 mol:1 mol, between about 1 mol: 10 mol and about 100 mol:1 mol, orbetween about 1 mol:1 mol and about 10 mol:1 mol. The ratio of theamount of dispase to the amount of growth factor(s) may be between about1 mol dispase: 1000 mol growth factor and about 1000 mol:1 mol, betweenabout 1 mol:100 mol and about 1000 mol:1 mol between about 1 mol:10 moland about 100 mol:1 mol, or between about 1 mol:10 mol and about 10mol:1 mol.

The treatment of growth factor(s) with proteases may be performed at atemperature between about 0° C. and about 40° C., about 4° C. and about40° C., or at about 37° C. or lower, in some embodiments. A growthfactor may be treated with collagenase for between about 15 minutes and3 days, about 30 minutes and about 3 days, about 30 minutes and about 48hours, about 30 minutes and about 24 hours, about 1 hour and about 18hours, about 1 hour and about 48 hours, about 2 hours and about 48hours, or about 2 hours and about 24 hours, in certain embodiments. Agrowth factor may be treated with trypsin or clostripain for betweenabout 5 minutes and 3 days, about 15 minutes and about 48 hours, about15 minutes and about 24 hours, or about 30 minutes and about 18 hours. Agrowth factor may be treated with dispase for between about 5 minutesand 2 days, about 15 minutes and about 24 hours, about 15 minutes andabout 18 hours, or about 15 minutes and about 8 hours. All theseconditions do not count the extra protein or peptide substrate in thetreatment mixture. Treatment may include mixing and/or incubation.Incubation may be performed under static or dynamic conditions, such aswith agitation, shaking, stirring, mixing, horizontal motion, rocking,and others.

In some embodiments, the protease may be a recombinant protein orpeptide fragment, a chemically synthesized protein or peptide fragment,or it may be extracted from a natural source and, optionally, modified,for example by being cleaved, heat-inactivated, chemically-modified, orother methods. In other embodiments, certain aspects of the enzymaticactivity of a protease may be inhibited or altered, and the proteasehaving altered activity may be added to one or more growth factors tochange the biological activity of the growth factor.

In additional embodiments, the activity of a protease may be modulatedbefore, during, or after treatment of at least one growth factor withthe protease. For example, the protease activity of a collagenase may besignificantly reduced before it is used to treat a growth factor. Inanother example, the activity of a protease may be modulated before,during, or after administering to cells. In certain embodiments, theactivity of a protease may be modulated by methods comprising heatinactivation, radiation inactivation, protease substrate neutralization,or chemical inhibition, among others. The chemical inhibitor used tomodulate the activity of metalloproteinases may be selected from metalchelating agents, such as EDTA; cysteine and serine protease inhibitorssuch as N-ethylmaleimide, phenylmethysulfonyl fluoride (PMSF), andleupeptin; classical metalloproteinase inhibitor, such asphosphoramidon, and bestatin; general protein inhibitor, such asα2-macroglobuli, or natural or synthetic tissue inhibitors ofmetalloproteinases (TIMPs), such as TIMP-1, TIMP-2, TIMP-3, TIMP-4; andother inhibitors.

The form of BMP-7 used in these methods can also be recombinant or canbe isolated from a variety of cellular or animal sources. In oneembodiment of the present invention, the BMP-7. In any form, that iscontacted with the at least one protease is a recombinant BMP-7. In onespecific embodiment, the recombinant BMP-7 is contacted with ametalloproteinase to generate the novel, truncated BMP-7 peptides of thepresent invention. In a more specific embodiment, the recombinant BMP-7is contacted with a collagenase to generate the novel, truncated BMP-7peptides of the present invention. In an even more specific embodiment,the recombinant BMP-7 that is contacted with a collagenase is arecombinant mature form of BMP-7. In a still more specific embodiment,recombinant mature BMP-7 is contacted with a collagenase and/orclostripain to generate the novel, truncated BMP-7 peptides of thepresent invention. In another specific embodiment, recombinant matureBMP-7 is contacted with a trypsin or dispase to generate the novel,truncated BMP-7 peptides of the present invention.

Once treated with at least one protease, in some embodiments, theresulting peptides are isolated and purified using routine methods inthe art. Examples of purification methods include, but are not limitedto size exclusion chromatography, high-performance liquidchromatography, ion exchange chromatography, electrophoresis, Westernblotting and subsequent processing of the membrane. In one embodiment ofthe present invention, the resulting peptide that is purified afterprotease treatment comprises the amino acid sequence of SEQ ID NO: 2 or3. In other embodiments, after being treated with a protease, theresulting peptides are not isolated or purified and are administered toa cell in the presence of the protease. In another embodiment, theresulting peptide is isolated or purified and then is administered to acell with a protease. In again another embodiment, after being treatedwith a protease, without being isolated or purified, the resultingpeptides are administered to a cell in the presence of the protease,then another protease is administered together.

The modified growth factor peptides of the present invention may also beprepared by synthetic methods using solid-phase synthetic techniques.The synthesized polypeptides may be re-natured into correct foldingpattern under appropriate conditions and bioactive dimmers can beinduced from monomers using appropriate buffers and conditions. Thecorrectly folded dimers maybe further separated from monomers usingappropriate column to increase the yield of functional bioactivefactors.

Regardless of method of preparation, be it recombinantly, enzymaticallyor synthetically the novel modified growth factors, as monomers ordimers, can be prepared as a composition. In one embodiment, thecomposition is a pharmaceutical composition. For example, one or morecofactors may be added to the truncated mature bioactive factor for thepresent invention to form a composition. Cofactors that may be addedinclude, but are not limited to, heparin, hyaluronic acid, afibronectin, an elastin, a laminin, albumin, a proteoglycan, collagen,gelatin, a divalent cation, calcium chlorid, zinc sulfate, magnesiumchloride, sodium bicarbonate, sodium chloride, sodium acetate, or sodiumphosphate. In some embodiments, a protein or a protein fragment may beadded as a cofactor to the modified growth factor peptides of thepresent invention.

The compositions, or pharmaceutical compositions, comprising the nucleicacid molecules or polypeptides typically comprise the nucleic acidmolecule or protein and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” or “pharmaceuticalcarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The nature of the pharmaceutical carrier or otheringredients will depend on the specific route of administration andparticular embodiment of the invention to be administered. Examples oftechniques and protocols that are useful in this context are, interalia, found in Remington: The Science and Practice of Pharmacy (2010),Lippincott, Williams & Wilkins. Examples of such pharmaceutical carriersor diluents include, but are not limited to, water, saline, Ringer'ssolution, dextrose solution and 5% human serum albumin. Liposomes andnon-aqueous vehicles such as fixed oils may also be used. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include oral and parenteral (e.g., intravenous,intradermal, subcutaneous, inhalation, transdermal (topical),transmucosal and rectal administration). Solutions or suspensions usedfor parenteral, intradermal or subcutaneous application can include, butare not limited to, a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents, antibacterial agents such as benzylalcohol or methyl parabens, antioxidants such as ascorbic acid or sodiumbisulfite, chelating agents such as ethylenediaminetetraacetic acid,buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablepharmaceutical carriers include physiological saline, bacteriostaticwater, Cremophor EL™ (BASF) or phosphate buffered saline (PBS). In allcases, the compositions must be sterile and should be fluid to theextent that easy syringeability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thepharmaceutical carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol ascorbic acid, thimerosal, and the like. In manycases, it may be desirable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., the modified growth factor) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation are vacuum drying and freeze-drying that yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutical carrier. They can be enclosed in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches or capsules. Oral compositionscan also be prepared using a fluid pharmaceutical carrier for use as amouthwash, wherein the compound in the fluid pharmaceutical carrier isapplied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like may contain any of the followingingredients, or compounds of a similar nature, such as but not limitedto a binder, such as microcrystalline cellulose, gum tragacanth orgelatin, an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel or corn starch, a lubricant such asmagnesium stearate or Sterotes, a glidant such as colloidal silicondioxide, a sweetening agent such as sucrose or saccharin, or a flavoringagent such as peppermint, methyl salicylate or orange flavoring.

In one embodiment, the active compounds are prepared with pharmaceuticalcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These compositions can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. It is especially advantageous to formulate oralor parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The pharmaceutical compositions can be included in a container, pack ordispenser together with instructions for administration.

The dosage of BMP-7 peptides will depend on the disease state orcondition to be treated and other clinical factors such as weight andcondition of the human or animal and the route of administration of thecompound. For treating human or animals, between approximately 0.005mg/kg of body weight to 500 mg/kg of body weight of the compound can beadministered. Therapy is typically administered at lower dosages and iscontinued until the desired therapeutic outcome is observed.

Methods of determining the dosages of compounds to be administered to apatient and modes of administering compounds to an organism aredisclosed in, for example, WO 96/22976. Those skilled in the art willappreciate that such descriptions are applicable to the presentinvention and can be easily adapted to it.

The proper dosage depends on various factors such as the type of diseasebeing treated, the particular composition being used and the size andphysiological condition of the patient. Therapeutically effective dosesfor the compounds described herein can be estimated initially from cellculture and animal models. For example, a dose can be formulated inanimal models to achieve a circulating concentration range thatinitially takes into account the IC₅₀ as determined in cell cultureassays. The animal model data can be used to more accurately determineuseful doses in humans.

The invention also relates to methods of altering intracellularsignaling of a cell, comprising contacting the cells with at least oneof the peptides of the present invention, wherein the cell possesses areceptor that specifically binds to the modified growth factors of thepresent invention. The peptides utilized in the methods can be inmonomeric, homodimeric or heterodimeric form. The specific binding ofthe modified growth factors to its receptor will, in turn, initiate theintracellular signaling cascade that is normally associated with theunmodified forms of the growth factor. For example, the “mature” form ofBMP-7 normally binds to its type I and/or type II receptors. Thereceptor phosphorylates cytoplasmic targets that include the Smad familyof proteins. Smads are a class of proteins that function asintracellular signaling effectors for the TGF-β superfamily of secretedpolypeptides. The activated BMP type I receptors will phosphorylate Smad1, Smad 5, and/or Smad 8. Phosphorylated Smad 1, 5, and 8 proteins, inturn, form a complex with Smad 4 and then translocate into the nucleusand interact with the transcription factors that regulate the expressionof target genes.

Accordingly, the present invention provides methods of stimulatingphosphorylation of Smad 1, Smad 5 and/or Smad 8 in a cell comprisingcontacting the cell(s) with at least one modified growth factor of theinvention. The peptides utilized in the methods can be in monomeric,homodimeric or heterodimeric form. The activity of the novel peptideswith respect to phosphorylating the Smad proteins may or may not bealtered relative to the normal, mature growth factor. For example, thenovel modified growth factors may increase or decrease phosphorylationof Smad 1, 5 and/or 8 compared to receptor binding of normal, maturegrowth factor. As another example, the novel modified growth factors maybind tighter or looser to its receptor (lower Kd) compared to receptorbinding of normal, mature growth factor. One of skill in the art canreadily determine if a particular protein is more or less phosphoylatedover control groups using well known techniques such as transcription ofreporter genes, ELISA assays, etc. Additional methods of the presentinvention comprise assessing the levels of Smad phosphorylation, forexample, Smad 1, Smad 5 or Smad 8, both before and after contacting thecell(s) with the novel peptides of the present invention and determiningthe increase or decrease of Smad phosphorylation in response to thenovel peptides of the present invention.

In another embodiment, the present invention provides methods ofstimulating promoter activity in a cell or population of cells, wherethe promoter is responsive to activated Smad complexes, with the methodscomprising contacting the cell(s) with at least one modified growthfactor peptide of the present invention. The peptides utilized in themethods can be in monomeric, homodimeric or heterodimeric form. One ofskill in the art would be aware of promoters that respond to activatedSmad complexes. See, for example, Massague (2000) Cell, 138:295-309. Theactivity of the novel peptides with respect to stimulatingSmad-responsive promoters may or may not be altered relative to thenormal, mature growth factor. For example, the novel modified growthfactors may increase or decrease activation of Smad-responsive promoterscompared to receptor binding of normal, mature growth factor. One ofskill in the art can readily determine if a promoter is more or lessactivated over control groups using well known techniques such astranscription of reporter genes, ELISA assays, etc. Additional methodsof the present invention comprise assessing the activity of aSmad-responsive promoter both before and after contacting the cell(s)with the novel peptides of the present invention and determining theincrease or decrease of the promoter in response to the novel peptidesof the present invention.

As used herein, “contacting,” when used in connection with the methodsof the present invention means bringing the novel peptides, inmonomeric, homodimeric or heterodimeric form, in proximity to the targetcells such that a specific binding event or a biological effect ispossible. Thus, contacting can include adding the novel peptides inculture medium and applying the culture medium to cells in culture.Contacting also encompasses transfecting a cell with at least one vectordescribed herein and allowing the cell to produce the modified growthfactor. Of course, contacting would also include administration of themodified growth factor peptides, or pharmaceutical compositions thereof,of the present invention to cells in an intact organism. Compositionsfor administering the novel peptides of the present invention have beendescribed herein.

As used herein, “administering” and “administer” are used to meanintroducing at least one compound comprising at least one novel modifiedgrowth factor peptide into a subject. The peptides utilized in theadministration methods can be in monomeric, homodimeric or heterodimericform. When administration is for the purpose of treatment, the substanceis provided at, or after the onset of, a symptom or condition in need oftreatment. The therapeutic administration of this substance serves toattenuate any symptom, or prevent additional symptoms from arising. Whenadministration is for the purposes of preventing a condition fromarising (“prophylactic administration”), the substance is provided inadvance of any visible or detectable symptom. The prophylacticadministration of the substance serves to attenuate subsequently arisingsymptoms or prevent symptoms from arising altogether. The route ofadministration of the compound includes, but is not limited to, topical,transdermal, intranasal, vaginal, rectal, oral, subcutaneousintravenous, intraarterial, intramuscular, intraosseous,intraperitoneal, epidural and intrathecal as previously disclosedherein.

Furthermore, the methods would also include coadministering one or moresubstances in addition to the novel peptides of the present invention.The term “coadminister” indicates that each of at least two compounds isadministered during a time frame wherein the respective periods ofbiological activity or effects overlap. Thus the term includessequential as well as coextensive administration of the compounds of thepreset invention. And similar to administering compounds,coadministration of more than one substance can be for therapeuticand/or prophylactic purposes. If more than one substance iscoadministered, the routes of administration of the two or moresubstances need not be the same.

The invention also relates to methods of promoting osteoinductivity,with the methods comprising contacting cells with at least one modifiedgrowth factor peptide of the present invention or compositions describedherein. As used herein, “osteoinductivity” can refer to causing cells todifferentiate into cells that are more osteoblast-like in phenotype, orthe term can refer to increasing the proliferation of osteoblasts, orboth. The cells, prior to contact with the modified growth factorpeptide(s) of the present invention, may be undifferentiated orpartially differentiated cells. The cells may be present in culture orin a tissue, organ or portion thereof or in an organism. Theosteoinductive activity of the novel peptides may or may not be altered,including but not limited to, enhanced activity, relative to the normal,mature growth factor.

The invention also relates to methods of promoting chondroinductivity,with the methods comprising contacting cells with at least one modifiedgrowth factor peptide of the present invention or compositions describedherein. As used herein, “chondroinductivity” can refer to causing cellsto differentiate into cells that are more chondrocyte-like in phenotype,or the term can refer to increasing the proliferation of chondrocytes,or both. The cells, prior to contact with the modified growth factorpeptide(s) of the present invention, may be undifferentiated orpartially differentiated cells. The cells may be present in culture orin a tissue, organ or portion thereof or in an organism. Thechondroinductive activity of the novel peptides may or may not bealtered, including but not limited to, enhanced activity, relative tothe normal, mature growth factor.

For example, the invention provides for growing and/or culturing cellsin the presence of one or more modified growth factor peptides orcompositions described herein. “Growing and/or culturing cells in thepresence of” includes traditional cell culture methods as well a placingcells in the presence of the modified growth factors in any setting,such as in natural or synthetic matrices or tissues. The cells may bemammalian, such as but not limited to human, bovine, porcine, murine,ovine, equine, canine, feline and others. In some embodiments, the cellsmay be mesenchymal stem cells, such as adipose-derived stem cells,embryonic stem cells, progenitor cells, differentiated cells,undifferentiated cells, and/or induced pluripotent stem cells.Appropriate cells may also include, but are not limited to cells of theectodermal lineage, cells of the mesodermal lineage, and cells of theendodermal lineage. Examples of cells of the ectodermal lineage includebut are not limited to keratinocytes, osteoblasts, chondrocytes,neurons. Examples of cells of the mesodermal lineage include but are notlimited to myoblasts, adipocytes, fibroblasts, endothelia cells, orstromal cells. Examples of cells of the endodermal lineage include butnot limited to epithelial cells of the auditory tube, the respiratorytract, such as trachea, bronchi, and alveoli of the lungs, thegastrointestinal tract, the urinary bladder and epithelial cells liningall glands. The cells may also be primary cells derived from tissues ororgans. Appropriate cell lines used in the present invention may includebut are not limited to mesenchymal cell lines, preosteoblastic celllines, osteoblastic cell lines, and chondroblastic cell lines. The cellsto which the modified growth factor peptides have been administered maybe placed directly into a tissue, organism or other setting such as amatrix, including, but not limited to, bone matrices.

In some embodiments, the cells may be derived from autologous orallogeneic sources. The cells may be differentiated cells includingchondrocytes, osteoblasts, osteoclasts, endothelial cells, epithelialcells, fibroblasts, and periosteal cells. Additionally, the cells may betotipotent, pluripotent, multipotent, progenitor, or adult somatic stemcells. The stem cells may be derived from embryos, placenta, bonemarrow, adipose tissue, blood vessel, amniotic fluid, synovial fluid,synovial membrane, pericardium, periosteum, dura, peripheral blood,umbilical blood, menstrual blood, baby teeth, nucleus pulposus, brain,skin, hair follicle, intestinal crypt, neural tissue, muscle. The stemcells may be derived from skeletal muscle, smooth muscle, and cardiacmuscle. The stem cells may be derived from genetic reprogramming ofmature cells, such as induced pluripotent stem cells (iPSCs).

Any cell described herewith may be cultured with one or more modifiedgrowth factors or compositions described herein for between about 15minutes and about 4 weeks, about 2 hours and about 2 weeks, about 2hours and about 1 week, about 2 hours and about 72 hours, about 24 hoursand about 72 hours, or about 24 hours and about 96 hours, at betweenabout 20° C. and about 40° C. or about 30° C. and about 37° C., in anatmosphere containing between about 1% CO₂ and about 10% CO₂ or about 4%CO₂ and about 6% CO₂, in certain embodiments. In some embodiments of thepresent invention, cells may be cultured in the presence of one or moremodified growth factors and (1) a tissue or an organ, (2) a matrix, or(3) a combination thereof. Cells that have been cultured in the presenceof one or more modified growth factors in a cell culture medium maysubsequently be applied to a matrix, a tissue, an organ or a combinationthereof, in certain embodiments.

In some embodiments, treated cells are cryopreserved. Cryopreservationagents may be used to preserve treated cells, in certain embodiments. Insome embodiments, treated cells are seeded onto a matrix, and thecell-seeded matrix may be preserved using at least one cryopreservationagent. At least one cofactor may be added to treated cells, in someembodiments. Cofactors that may optionally be used are heparin,hyaluronic acid, a fibronectin, an elastin, a laminin, a proteoglycancollagen, or gelatin, among others. In certain embodiments, a divalentcation, calcium chloride, zinc sulfate, magnesium chloride, heparin,sodium bicarbonate, sodium chloride, or sodium phosphate may be added ascofactor to treated cells. In some embodiments, a protein or a proteinfragment may be a cofactor that is added to treated cells. In furtherembodiments, a protease described herein may be a cofactor that is addedto treated cells.

In certain embodiments, treated cells that have been cryopreserved areoptionally revived at a temperature between about 10° C. and about 37°C. before being applied to a tissue or an organ defect, a tissue, anorgan, a matrix, or a mixture of two or more of these. In certainembodiments, treated cells may be combined with one or more body fluid,for example blood, platelet-rich plasma, plasma, bone marrow, and cordblood, among others, and an isotonic, hypotonic, or hypertonic solution,for instance saline, or Lactated ringer solution, and others, beforethey are used to treat a tissue or an organ defect. In some embodiments,treated cells may be applied to a tissue or an organ defect by injectingor inserting the cells between tissues or organs, or placing the cellson top of the defect. Modified growth factors may be administered tocells in vitro, in vivo, or in situ, in some embodiments. The modifiedgrowth factors may be administered to cells that are in tissue or organor that have been isolated from tissues or organs, in some embodiments.In certain embodiments, at least one modified growth factor orcompositions described herein may be administered to cells in (1) a cellculture medium, (2) a tissue or an organ, (3) a matrix, or (4) acombination of two or more of these.

There are a variety of osteoblast or chondrocyte differentiation markersthat can be measured to assess osteoinductivity or chondroinductivity,respectively. For example, cells express alkaline phosphatases duringthe early stages of differentiation toward osteoblast lineages.Therefore, in vitro alkaline phosphatase assays may be used to evaluateosteoinductivity in cells contacted with the modified growth factorpeptide(s) of the present invention. The ability of the modified growthfactor peptide(s) of the present invention to stimulate or induce thealkaline phosphatase expression in an otherwise non-bone forming cells,such as myoblast (C2C12 cells), would indicate that the modified growthfactor peptide(s) of the present invention has osteoinductive activity.In these assays, cells cultured without added growth factors of any kindand without added modified growth factor peptide(s) of the presentinvention are used as negative controls to show that the baselinealkaline phosphatase expression on non-bone forming cells. The baselineof the osteoblastic markers in the negative control need not be zero,meaning that the cells in the negative control group may have at leastsome level of phenotypic marker(s) associated with osteoblasts.Accordingly, an “osteoinductive” peptide of the present invention wouldsimply cause an increase in the predetermined osteoblastic markers inexperimental cells over control. Similarly, chondrocyte markers,including but not limited to type X collagen, type II collagen, Sox 9,Aggrecan. Matrilin-1 and CEP-68, to name a few, can be used to assesschondroinductive potential.

Moreover, osteoinductivity and/or chondroinductivity may be determinedin tissue culture by investigating the ability of the modified growthfactor peptide(s) of the present invention to differentiate or induceosteoblast phenotype or chondrocyte phenotype in cultured cells, such asprimary cells, cell lines, or explants. For example, the cells maydisplay increased production of a marker characteristic of osteoblastsand/or chondrocytes, such as alkaline phosphatase, etc. For example, theosteoinductive or chondroinductive potentials of the modified growthfactors may be more than 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 times greater for the modified growth factors compared to theunmodified growth factors, in another example, the osteoinductive orchondroinductive potentials of the modified growth factors may be morethan 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500 or even 1000 timesgreater for the modified growth factors compared to the unmodifiedgrowth factors. Of course, this indicates that lower concentrations ofmodified growth factor, compared to unmodified growth factor arerequired to achieve the same effects.

Osteoinductivity or chondroinductivity, for assessing the bone orcartilage forming potential induced by the modified growth factorpeptide(s) of the present invention in a location such as muscle, mayalso be evaluated using a suitable animal model. For example,intramuscular implantation into a rodent has been used as a model toassess osteoinductive activity of bioactive factors.

The invention also relates to methods of promoting proliferation ormaintaining the differentiated state of osteoblasts and/or chondrocytesand/or any cell type disclosed herein comprising administering to theosteoblasts or chondrocytes the modified growth factor peptides orcompositions described herein. The proliferative activity of the novelpeptides may or may not be altered, including but not limited to,enhanced activity, relative to the normal, mature growth factor.

Mitogenicity may be assessed by investigating cell proliferation inducedby the modified growth factor peptides using various in vitro assaysthat measure metabolic activity, such as MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay,alamarBlue® assay, and others. The alamarBlue® assay uses anon-cytotoxic reduction-oxidation indicator to measure cell metabolicactivity, making it a nondestructive assay for assessing the mitogenicactivity of the modified growth factors. Proliferation can also beassessed by measuring DNA quantification, such as by using a PicoGreen™DNA assay, radioactive labeling of DNA synthesis, such as[^(3H)]thymidine labeling or BrdU incorporation. Proliferation can alsobe assessed via manual cell counting, such as using a trypan bluehemacytometer.

The invention also relates to methods of increasing a cellular growthfactor activity comprising administering to the cell at least oneprotease and the growth factor. The protease is a protease describedherein, including, but not limited to, collagenase, clostripain,dispase, trypsin, BMP-1, MMP-13, and a mixture thereof. In someembodiments, the growth factor comprises the modified growth factordescribed herein. The cell is a cell described herein, including, butnot limited to, differentiated cells, adult stem cells, progenitorcells, and induced pluripotent stem cells. As described previously, themodification of the growth factors as disclosed herein also enhances theactivity of BMP-7. In certain embodiments, administering the proteasealong with the modified growth factor described herein further enhancesthe activity of the modified growth factor, such as an osteoinductive orchondroinductive activity.

In some embodiments, the addition of the protease described herein intothe composition enhances stability of the growth factor in the samecomposition. As described previously, the modification of the growthfactors as disclosed herein also enhances the stability of the growthfactor. In certain embodiments, the addition of the protease into thecomposition comprising the modified growth factor further enhances thestability of the modified growth factor.

In additional embodiments, the composition administered to a cell inincreasing a cellular growth factor activity comprises two or moreprotease. In some embodiments, a second protease can be mixed into thecomposition comprising the protease and the growth factor or can beadministered separately from the composition comprising the protease andthe growth factor.

The invention further relates to tissue or organ repair or regenerationcompositions comprising at least one modified growth factor peptide anda tissue, an organ, a matrix and/or a mixture of two or more thereof.The tissue or organ repair composition may comprise demineralized boneand homogenized connective tissue.

An implantable biocompatible matrix for use with the compositionsdescribed herein can function as a suitable delivery or support systemfor the modified growth factor peptides. A biocompatible matrix shouldbe non-toxic, non-eliciting or stimulating severe inflammatory responseor immunological rejections, and devoid of other undesired reactions atthe implantation site. Suitable matrices may also provide for release ofthe modified growth factor peptides, for example, to promote a slow,sustained release over time at the implantation site. In one embodiment,the matrix is a bone matrix or cartilage. In addition to its common,ordinary meaning, “administering to the matrix” includes embedding intothe matrix or matrices host cells capable of producing at least onemodified growth factors peptide. In another embodiment, the inventionprovides for methods of treating cells with at least one modified growthfactor peptide or compositions described herein and implanting thesetreated cells into a matrix, such as, but not limited to a bone matrixor cartilage.

Suitable matrices include, but are not limited to, porous scaffolds intowhich bone cells or progenitor cells may migrate. Osteogenic orchondrogenic cells, i.e., cells involved in the process of deposition ofnew bone material or cartilaginous material, respectively, can oftenattach to such porous matrices, which can then serve as scaffolding forbone and cartilage tissue growth. For certain applications, the matrixshould have sufficient mechanical strength to maintain its threedimensional structure and help support the immobilization of the bonesegments being united or grafted together. Porous matrices which providescaffolding for tissue growth can accelerate the deposition of new boneor the rate of bone growth and are said to be “osteoconductive.”Osteoconductive matrices are especially useful in the pharmaceuticalcompositions described herein. Porous matrices which provide scaffoldingfor tissue growth can accelerate the deposition of new cartilage or therate of cartilage growth and are said to be “chondroconductive.”Osteoconductive matrices are especially useful in the pharmaceuticalcompositions described herein. Chondroconductive matrices are especiallyuseful in the pharmaceutical compositions described herein. Theosteoinductive or chondroinductive activity of the novel peptides may ormay not be altered, including but not limited to, enhanced activity,relative to the normal, mature growth factor. Thus, the osteoconductiveor chondroconductive activity of the treated matrices of the presentinvention may be enhanced compared to matrices not treated with modifiedgrowth factor. Of course, the matrices are considered to beosteoconductive or chondroconductive if cells within the matrix begin todifferentiate into more osteoblast-like or chondrocyte-like appearing orfunctional cells, respectively.

Matrices can be derived from natural sources or they can be synthetic ora mixture of both. Matrices from natural sources may also comprisenatural polymers, including, but not limited to, collagen, hyaluronicacid, alginate, albumin, fibrinogen-fibrin, chitosan, elasin, laminin,connective tissues, cortical or cancellous bone, demineralized ormineralized bone, fascia lata, dermis, muscle, ligament, tendon, amixture thereof, and mixture of reconstituted tissue. Matrices fromsynthetic sources refer to any material not produced by livingorganisms, which may include, not limited to, the synthetic materialmade up of organic components, inorganic components, or a mixturethereof. In some embodiments, a synthetic matrix may comprise an organicsynthetic polymer, such as poly(lactic-co-glycolic acid),polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA),polyhydroxybutyrate (PHB), Poly(ethylene glycol) (PEG), poly(ethyleneoxide) (PEO)), and others. In some embodiments, a tissue, an organ, ormatrix comprising at least one of alginate, chitosan, collagen, gelatin,hyaluronic acid, a fibronectin, an elastin, a laminin, and aproteoglycan may be employed. In certain embodiments, a matrixcomprising inorganic components, such as hydroxyapatite, calciumsulfate, octacalcium phosphate, calcium phosphate macroporous calciummetaphosphate ceramic, β-tricalcium phosphate, metal, metal alloy, andothers, ma be used. A matrix used in certain embodiments of the presentinvention may be prepared by demineralizing, decellularizing ordevitalizing a tissue or an organ and cells ma be seeded onto thematrix.

In some embodiments, at least one modified growth factor or compositionsdescribed herein may be applied to the matrix and may be incubated atconditions permitting the generation of a treated matrix. In someembodiments, incubation may be carried out at about 40° C. or lower, orbetween about 10° C. and about 37° C., or about 20° C. and about 37° C.Incubation may be carried out for between at least about 2 minutes andabout 120 minutes, about 3 minutes and about 100 minutes, about 4minutes and about 80 minutes, about 5 minutes and about 60 minutes, andabout 5 minutes and about 30 minutes in certain embodiments. Incubationmay be performed under static or dynamic conditions, such as withagitation, shaking, stirring, mixing, horizontal motion, rocking, andothers.

In some embodiments of the present invention, a matrix may belyophilized before at least one modified growth factor or compositionsdescribed herein are administered to it. In certain embodiments, one ormore modified growth factors may be administered to a matrix, and thetreated matrix may be subsequently lyophilized. The lyophilized, treatedmatrix can then be rehydrated before it is used. Further, the cells canbe seeded onto the matrix before implantation.

Examples of suitable osteoconductive or chondroconductive matricesinclude but are not limited to, collagen (e.g. bovine dermal collagen),fibrin, calcium phosphate ceramics (e.g., hydroxyapatite and tricalciumphosphate), calcium sulfate, guanidine-extracted allogenic bone andcombinations thereof. A number of suitable matrices are commerciallyavailable, such as Collograft™ (Collagen Corporation), which is amixture of hydroxyapatite, tricalcium phosphate and fibrillar collagen,and Interpore™ (Interpore International), which is a hydroxyapatitebiomatrix formed by the conversion of marine coral calcium carbonate tocrystalline hydroxyapatite.

A number of synthetic biodegradable polymers can serve asosteoconductive or chondroconductive matrices with sustained releasecharacteristics. Descriptions of these polymers can be found inBehravesh (1999) Clinical Orthopedics 367, S118 and Lu (2000) PolymericDelivery Vehicles for Bone Growth Factors in Controlled Drug Delivery:Designing Technologies for the Future, Park and Mrsny eds., AmericanChemical Society. Examples of these polymers include polyα-hydroxyesters such as polylactic acid/polyglycolic acid homopolymers andcopolymers, polyphosphazenes (PPHOS), polyanhydrides and poly(propylenefumarates).

Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are wellknown in the art as sustained release vehicles. The rate of release canbe adjusted by the skilled artisan by variation of polylactic acid topolyglycolic acid ratio and the molecular weight of the polymer (seeAnderson (1997) Adv. Drug Deliv. Rex. 28:5. The incorporation of PEGinto the polymer as a blend to form microparticle matrices allowsfurther alteration of the release profile of the active ingredient (seeCleek (1997) J. Control Release 48, 259). Ceramics such as calciumphosphate and hydroxyapatite can also be incorporated into theformulation to improve mechanical qualities.

In one embodiment, the matrices used in the compositions and methods ofthe present invention are bone matrices. As used herein, a bone matrixis a matrix derived from or including elements of natural bone. In someembodiments, the natural bone is mineralized, partially demineralized,demineralized, cancellous, cortical, or cortical cancellous bone. Thebone matrices used herein may or may not include additional syntheticcomponents not typically found in bone tissue. Other embodiments includecompositions and methods utilizing a matrix derived from cartilage,other soft tissues such as the dermis, connective tissue, fascia, smallintestine submucosa, serous membrane, pericardium, tendon, ligament,muscle, adipose tissue, myelin, blood vessels, base membrane, amnioticmembrane and others. A matrix prepared from hyaline cartilage,fibrocartilage or elastic cartilage, may be employed in someembodiments. A matrix may be prepared from hyaline cartilage found inthe condyle, tibial plateau, femoral head, humeral head, costalcartilage, or fibrocartilage found in intervertebral discs, or elasticcartilage found in the epiglottis or ear. In certain embodiments, amatrix derived from natural sources that has been optionally cleaned,disinfected, chemically modified, decellularized, particulated,homogenized, lyophilized, gamma ray irradiated, and/or plasticized maybe used. Any of the matrices used herein may or may not includeadditional synthetic components not typically found in such tissue.

In one specific embodiment, the bone matrices or cartilage matrices maybe demineralized or decellularized, respectively. Examples ofdemineralized matrices and methods of making are described in U.S. Pat.Nos. 6,189,537 and 6,305,379.

The matrix, tissue, or organ used in certain embodiments of the presentinvention may be in the form of a powder, particulates, sheets, fibers,gels, putties, paste, blocks, cylinders, sponges, meshes, films, slices,curls, flakes, or wedges, among others. In certain embodiments of thepresent invention the matrix, tissue, or organ comprising the modifiedgrowth factor peptide(s) may be in the form of a powder, fibers, putty,or a sponge. In further embodiments, the sponge can include, forexample, the implant having sponge-like structures disclosed in theco-pending, commonly-assigned patent application PCT/US09/04556 entitled“Composition for a Tissue Repair Implant and Methods of Making the Same”filed on Aug. 7, 2009. The treated matrices can be used in any of themethods of the present invention.

The invention also relates to methods of increasing or promotingosteogenesis or chondrogenesis in cells. The methods may comprisetreating the cells in matrices with at least one of the modified growthfactor peptides or compositions described herein. As used herein,“osteogenesis” is the deposition new bone material or formation of newbone, including, but not limited to, intramembranous osteogenesis andendochondral osteogenesis. As used herein, “chondrogenesis” is thedeposition new cartilage material or formation of new cartilage. Theosteogenic or chondrogenic inducing activity of the novel peptides mayor may not be altered, including but not limited to, enhanced activity,relative to the normal, mature growth factor. The cells to which themodified growth factors may be administered include cells in any tissuein which bone or cartilage formation is desired, such as, but notlimited to, bone, cartilage, ligament, muscle, etc.

The invention also relates to methods of treating a tissue or organdefect or injury, for example, a musculoskeletal, dental or soft-tissuedefect or injury, in an animal comprising administering (1) cellscultured in the presence of one or more modified growth factors orcompositions described herein and/or (2) one or more modified growthfactors or compositions described herein to the tissue or organ defect.

The invention further relates to methods of treating a tissue or anorgan defect or injury, for example a musculoskeletal, dental orsoft-tissue defect, in an animal by applying a treated matrix to thedefect, and application to the defect may be accomplished by injectingthe treated matrix into the defect, inserting the treated matrix betweentissue or organ, or placing the treated matrix on top of the defect. Thepresent invention is also directed to treating a defect or injury in anorgan in a similar manner. At least one cofactor may be added to atreated matrix, such as a matrix comprising a modified growth factor, insome embodiments.

In yet another embodiment, cells may be seeded onto a treated matrix.The cells seeded on the treated matrix can be any cell, such as but notlimited to, osteoblasts, chondrocytes, progenitor cells, and stem cellsdisclosed herein or otherwise known in the art. The seeded cells may beallowed to proliferate and possibly attach to the matrix. Methods ofseeding cells onto matrices, such as collagen, are well known in theart. Alternatively, cells may first be treated with at least onemodified growth factor or compositions described herein and the treatedcells may then be seeded onto a treated or untreated matrix.

Any of the methods of the present invention can be performed invirtually any setting, such as an in vivo, ex vivo, in situ or in vitrosetting. For example, methods of promoting osteogenesis in cells may beperformed in cell culture, or may be performed in an intact organism.Moreover, any combination of any two or more of any of the embodimentsdescribed herein are contemplated.

While the invention has been described and illustrated herein byreferences to various specific materials, procedures and examples, it isunderstood that the invention is not restricted to the particularcombinations of material and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplars, only, with thetrue scope and spirit of the invention being indicated by the followingclaims. All references, patents and patent applications referred to inthis application are herein incorporated by reference in their entirety.

The following examples are illustrative and are not intended to limitthe scope of the invention described herein.

Example 1—Preparation and Sequencing of Collagenase-Modified BMP-7

The mature rhBMP-7 was treated with collagenase in a microcentrifugetube at a molarity ratio of between about 1:10 and about 100:1 andincubated overnight at 37° C. The treated mature rhBMP-7 was furtherpurified by reverse phase HPLC using a C-18 column. The collectedfractions from HPLC were tested for osteoinductive potential using an invitro alkaline phosphatase assay. The fractions that showedsignificantly high alkaline phosphatase activities were furtherseparated by SDS-PAGE gel electrophoresis and blotted onto PVDFmembrane. Proteins on the PVDF membrane were stained by coomassie blueand the related bands were cut and sent for N-terminal sequencing.

In the Coomassie blue stained PVDF membrane, the fraction 43 sample fromthe collagenase-treated rhBMP-7 showed clean bands at around 15-16 kDa.

Ten amino acids of the N-terminus of the collagenase treated maturerhBMP-7 were detected using Edman degradation chemistry with an ABIProcise® 494 sequencer. The deduced amino acid sequence of theN-terminus of the treated rhBMP-7 was taken to be ENSSSDQRQA, which isthe first 10 amino acids of SEQ ID NO: 3 herein. This deduced N-terminusof the treated rhBMP-7 matches the untreated mature rhBMP-7 sequenceexcept, indicating that the collagenase cleaved the mature BMP-7 afterthe first 27 amino acids. Western blot detected untreated andcollagenase-treated mature rhBMP-7, and the treated rhBMP-7 was about2-3 kDa smaller than the untreated rhBMP-7.

Example 2—Osteoinductive Potential of Collagenase-Modified BMP-7

Alkaline phosphatase is one of the distinctive biological orbiologically-derived indicators of osteoinductivity. The AP assaymeasures the product para-nitrophenol (pNP) at a wavelength of 405 nmafter 60 minutes of incubation of the substrate para-nitrophenylphosphate (pNPP) with a cell lysate at 37° C. The alkaline phosphataseactivities induced by the HPLC fractions from Example 1 are shown inFIG. 1. The alkaline phosphatase activity induced by the fraction 43from the collagen-treated rhBMP-7 was significantly higher than thealkaline phosphatase activity induced by other HPLC fractions.

Example 3—Dose Responsiveness of Modified rhBMP-7

C2C12 cells (ATCC CRL-1772) were seeded at a density of 25,000 cells/cm²in 24-well plates on day one. Collagenase and various concentrations ofrhBMP7 were mixed in microcentrifuge tubes at molarity ratios betweenabout 1:10 and about 1000:1 and incubated at 37° C. overnight. Thecollagenase-treated rhBMP7 or untreated rhBMP7, was introduced into eachwell of the C2C12 cell seeded 24-well plate on day two. The finalconcentrations of rhBMP7 were 5 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, 100ng/mL, and 200 ng/mL in each well. After 3 days of incubation at 37° C.and 5% CO₂, cells were collected from culture plates and cell lysateswere prepared for AP assays and BCA total protein assays.

The results of the in vitro AP assay are shown in FIG. 2. When cellswere cultured with treated or untreated rhBMP7 at a concentration of 10ng/mL or less, the differences in alkaline phosphatase activity werenegligible. At concentrations of 25 ng/mL or higher, significantdifferences were found between collagenase-treated rhBMP7 groups anduntreated rhBMP7 groups. At 50 ng/mL in cell culture, the alkalinephosphatase activity induced by collagenase-treated rhBMP7 was about 80times higher than that induced by untreated rhBMP7.

Example 4—Preparation and Sequencing of Trypsin-Modified BMP-7

rhBMP-7 was incubated with trypsin overnight at 37° C. Trypsin andrhBMP-7 were used at a molarity ratio between about 1:50 and about100:1. The treated mature rhBMP-7 was separated by SDS-PAGE gelelectrophoresis followed by protein transferring onto PVDF membrane. Theproteins on PVDF membrane were stained by Coomassie blue and the relatedbands were cut for N-terminal sequencing using Edman degradationchemistry with an ABI Procise 494 sequencer. Proteins on PVDF membranewere also detected by Western Blot using antibody against C-terminus ofrhBMP-7.

By comparing the trypsin treated mature rhBMP-7 bands with the untreatedmature rhBMP-7 control bands, extra band at around 14-16 kDa were cutfor N-terminal sequencing. Ten amino acids of the N-terminus of thetrypsin treated mature rhBMP-7 were detected using Edman degradationchemistry with an ABI Procise® 494 sequencer. The deduced amino acidsequence of the N-terminus of the treated rhBMP-7 was QACKKHELYV. TheN-terminus of the trypsin-treated rhBMP-7 matches the untreated maturerhBMP-7 sequence, indicating that the trypsin cleaved the mature BMP-7after the first 35 amino acids. The Western blot detected a band fortrypsin treated rhBMP-7 group, which is about 2-3 kDa smaller than theband for untreated mature rhBMP-7.

Example 5—Osteoinductive Potential of Trypsin-Modified BMP-7

C2C12 cells were cultured as described above. Trypsin and rhBMP7 weremixed in a microcentrifuge tube at molarity ratios between about 1:50and about 100:1 and incubated at 37° C. overnight. The trypsin-modifiedrhBMP7 or unmodified rhBMP7 was introduced into each well of the C2C12cell seeded 24-well plate on day two. After 3 days of incubation at 37°C. and 5% CO₂, cells were collected from culture plates and cell lysateswere prepared for AP assays and BCA total protein assays.

The results of the in vitro AP assay are shown in FIG. 3. The alkalinephosphatase activity induced by trypsin-modified rhBMP7 was about 1.5times higher than that induced by unmodified rhBMP7.

Example 6—Osteoinductive Potential of Modified rhBMP-7 with or without aProtease

C2C12 cells were cultured as described above. Collagenase and rhBMP-7are mixed in microcentrifuge tubes at a molarity ratio of between about1:10 and about 100:1 and are incubated overnight at 37° C. The treatedmature rhBMP-7 is aliquoted with or without further HPLC purificationdescribed above. The aliquot with further HPLC purification (purifiedmodified BMP-7) is quantified by ELISA. The aliquot without further HPLCpurification (non-purified modified BMP-7) retained the collagenase inthe aliquot. The aliquots are used to test the effect of treated maturerhBMP-7 on osteogenic potential of C2C12 myoblasts.

Each of the rhBMP-7 control, and the purified modified rhBMP-7, and thenon-purified modified rhBMP-7 at the same concentration is introducedinto wells of the C2C12 cell seeded 24-well plate. C2C12 cells culturedin media alone (without addition of rhBMP-7 or modified rhBMP-7) areused as a negative control.

After 3 days incubation at 37° C., 5% CO₂, the AP assay as describedabove is performed. The alkaline phosphatase activity of cells culturedwith the purified modified rhBMP-7 or the non-purified modified rhBMP-7are significantly higher than that of cells cultured with non-treatedrhBMP-7 control. The alkaline phosphatase activity of cells culturedwith non-purified modified rhBMP-7 is also significantly higher thanthat of cells cultured with purified modified rhBMP-7. The resultsdemonstrate a significant increase in the alkaline phosphatase activityby administering the non-purified modified rhBMP-7 compared toadministering the purified modified rhBMP-7.

Further, with another set of C2C12 cells cultured as described above,the purified modified rhBMP-7 is introduced into wells of the C2C12 cellalong with 138 ng/mL of active dispase or inactive dispase, or 100 ug/mLcollagenase or inactive collagenase. The AP assay as described above isperformed, and the results show that adding active or inactive dispaseor collagenase to the purified modified rhBMP-7 samples also increasethe alkaline phosphatase activity.

In addition, rhBMP-7 treated by clostripain, dispase, and the mixturethereof, without further purification, also increase the alkalinephosphatase activity compared to non-modified rhBMP-7 control. Inaddition, rhBMP-7 treated by BMP1 or MMP13, without furtherpurification, also increase the alkaline phosphatase activity comparedto non-modified rhBMP-7 control.

Example 7—Osteoinductive Potential of Modified rhBMP-7 with or withoutan Additional Protease

Dispase and rhBMP-7 were mixed in microcentrifuge tubes at a molarityratio between about 1:100 and about 1000:1 and incubated at 37° C. fromabout 15 minutes to about 24 hours.

C2C12 cells were cultured as described above. The non-treated rhBMP-7control, dispase modified rhBMP-7 without further purification, ordispase modified rhBMP-7 with the addition of 100 ug/mL collagenase inculture media were introduced into wells of the C2C12 cell seeded24-well plate on day two, C2C12 cells cultured in media alone (withoutaddition of rhBMP-7 or treated rhBMP-7) were used as a negative control.After 3 days of incubation at 37° C./5% CO₂, cells were collected fromculture plates and cell lysates were prepared for AP assays and BCAtotal protein assays as described above.

The results of the in vitro AP assay are shown in FIG. 4. The alkalinephosphatase activity of cells cultured with dispase modified rhBMP-7 wasabout 141% higher than that of cells cultured with non-treated rhBMP-7control, while the alkaline phosphatase activity of cells cultured withdispase modified rhBMP-7 with the addition of collagenase in mediafurther increased 116% compared to the dispase modified rhBMP-7 group.The alkaline phosphatase activity of cells cultured with dispasemodified rhBMP-7 with the addition of collagenase in media was about421% higher than that of cells cultured with non-treated rhBMP-7control.

Example 8—Osteoinductive Potential of Modified rhBMP-7 with a Protease

C2C12 cells (ATCC CRL-1772) are cultured as described above. Collagenaseand rhBMP-7 are mixed in microcentrifuge tubes at a molarity ratio ofbetween about 1:10 and about 100:1 and incubated overnight at 37° C.with agitation. The treated mature rhBMP-7 is aliquoted, and thealiquots are used to test the effect of treated mature rhBMP-7 onosteogenic potential of C2C12 myoblasts as follows.

For the osteoinductive potential assessment, base media of DMEMcontaining 1% FBS, 50 μg/mL of ascorbic acid and 10 mM ofβ-glycerolphosphate is used as a control group. The base media controlwith the addition of 50 ng/mL of rhBMP-7 is used as the BMP-7 positivecontrol group. The base media control with the addition of 50 ng/mL ofmodified rhBMP-7 without the purification is used as the test group. Themedia are added into C2C12 cells seeded on chamber slides and changedevery 3-4 days.

After several days of incubation at 37° C., 5% CO₂, photographs are alsotaken for cell cultures in each group. The nodule formation of C2C12cells appear only in modified rhBMP-7 group, but not in the other twocontrol groups. After more days of incubation, the media is removed fromchamber slides and the slides are stained with Alizarin Red S. Alizarinred S is used to identify calcium-rich deposits by cells in culture ortissue sections, which indicates the effect of osteoinductive materialon osteogenic potential of cells.

For the base media control group, no positive Alizarin red S staining isfound at any tested time points. For the rhBMP-7 control group, someAlizarin red S staining is detected after 6 or 12 days of incubation.For the modified rhBMP-7 group, after 6 days of incubation, significantamount of nodules stain positive by Alizarin red S. After 12 days ofincubation, more Alizarin red S positively stain nodules of larger sizeare observed in C2C12 cells cultured with modified rhBMP-7 than that inC2C12 cells cultured with unmodified rhBMP-7. This data suggests thatmodified rhBMP-7 has a significantly greater effect on osteogenicpotential of C2C12 myoblasts as compared to the unmodified maturerhBMP-7 control.

Example 9—Stability of Modified rhBMP-7 with or without a Protease inCell Culture

C2C12 cells are cultured, and purified and non-purified modified rhBMP-7are prepared as described in Example 3 above. The purified modifiedrhBMP-7, the non-purified modified rhBMP-7, or the untreated maturerhBMP-7 at the same concentration is introduced into each well of theC2C12 cell seeded 24-well plate on day two. C2C12 cells cultured withoutaddition of rhBMP-7 or modified rhBMP-7 are used as a negative control.After incubation at 37° C., 5% CO₂ for different durations (24 hr, 48hr, or 72 hr), spent culture media are collected from these original24-well plates and transferred into new 24-well plates containing freshC2C12 cells seeded the previous day, and the new 24-well plates areincubated at 37° C., 5% CO₂ for another 3 days. Cells from the original24-well plates and new 24-well plates are collected at the end of eachculture period, and cell lysates are prepared for AP assays and BCAtotal protein assays.

For the control groups wherein C2C12 cells are cultured for three daysin untreated rhBMP-7 spent media collected after 24 hrs, 48 hrs, and 72hrs, the alkaline phosphatase activity is reduced as compared to that ofcells cultured for three days with fresh rhBMP-7 media. For theexperimental groups in which C2C12 cells are cultured for three days inpurified modified rhBMP-7 spent media collected after 24 hrs, 48 hrs,and 72 hrs, the alkaline phosphatase activity is also reduced less thanfor the control group. For the experimental groups in which C2C12 cellsare cultured for three days in non-purified modified rhBMP-7 spent mediacollected after 24 hrs, 48 hrs, and 72 hrs, however, the alkalinephosphatase activity is maintained at a similar or higher level ascompared to that of cells cultured for three days with freshnon-purified modified rhBMP-7 media. This demonstrates that the presenceof a protease can further enhance the stability of modified BMP-7 inculture media.

Example 10—Stability of Modified rhBMP-7 with or without a Protease onShelf

Purified and non-purified modified rhBMP-7 is prepared as described inExample 3 above. Aliquots of the purified and non-purified modifiedrhBMP-7 at the same volume and concentration are incubated at 37° C. fordifferent durations (2 days to 2 weeks). After each time point, onealiquot from each group is transferred into −20° C. freezer until thelast time point is completed. The aliquots of purified modified rhBMP-7or non-purified modified rhBMP-7 from different time points areintroduced into each well of the C2C12 cell cultured as described above.C2C12 cells cultured without the addition of rhBMP-7 or collagenase areused as a negative control. After 3 days incubation at 37° C., 5% CO₂,the AP assay as described above is performed.

The alkaline phosphatase activity of cells cultured with purifiedmodified rhBMP-7 on shelf (37° C.) for 7 days or 14 days is reduced ascompared to that of cells cultured with purified modified rhBMP-7 onshelf (37° C.) for 2 days. The alkaline phosphatase activity of cellscultured with non-purified modified rhBMP-7 on shelf (37° C.) for 7 daysor 14 days is reduced less than that of cells cultured with purifiedmodified rhBMP-7. This demonstrates that the presence of a protease canfurther enhance the stability of modified BMP-7 on shelf.

What is claimed is:
 1. A composition comprising at least onepharmaceutically acceptable carrier, at least one protease that is notbone morphogenetic protein-1 (BMP-1) or a cysteine protease, and apeptide consisting of the amino acid sequence of SEQ ID NO: 3, whereinthe peptide has osteoinductive activity or chondroinductive activity. 2.A composition comprising at least one pharmaceutically acceptablecarrier, at least one protease that is not bone morphogenetic protein-1(BMP-1) or a cysteine protease, and a peptide consisting of the aminoacid sequence of SEQ ID NO: 3, wherein the peptide has osteoinductiveactivity or chondroinductive activity, and wherein the molar ratio ofthe at least one protease to the peptide is between 1:1000 and 1000:1.3. The composition according to claim 1, wherein the at least oneprotease is selected from the group consisting of collagenase,clostripain, dispase, trypsin, MMP-13 (matrix metalloproteinase-13), anda mixture thereof.
 4. The composition according to claim 1, wherein theconcentration of the peptide is between 10 ng/mL and 200 ng/mL.
 5. Thecomposition according to claim 1, wherein the concentration of thepeptide is between 10 ng/mL and 100 ng/mL.
 6. The composition accordingto claim 1, wherein the concentration of the peptide is between 10 ng/mLand 50 ng/mL.
 7. The composition according to claim 1, wherein theconcentration of the peptide is between 10 ng/mL and 25 ng/mL.
 8. Thecomposition according to claim 1, wherein the pharmaceuticallyacceptable carrier is selected from the group consisting of saline,Ringer's solution, dextrose solution, 5% human serum albumin, dispersionmedia, coatings, antibacterial and antifungal agents, and isotonic andabsorption delaying agents.
 9. A composition comprising apharmaceutically acceptable carrier and a peptide consisting of theamino acid sequence of SEQ ID NO:
 3. 10. A method of promotingdifferentiation of cells into osteoblasts or chondrocytes, the methodcomprising treating the cells with the composition of claim 1 in anamount effective to promote the differentiation, wherein the cells areprogenitor cells, adult stem cells, or induced pluripotent stem cells.11. The method of claim 10, wherein the cells are progenitor cells oradult stem cells.
 12. The method of claim 11, wherein the progenitorcells or the adult stem cells are derived from placenta, bone marrow,adipose tissue, blood vessel, amniotic fluid, synovial fluid, synovialmembrane, pericardium, periosteum, dura, peripheral blood, umbilicalblood, menstrual blood, baby teeth, nucleus pulposus, brain, skin, hairfollicle, intestinal crypt, neural tissue, or muscle.
 13. The method ofclaim 10, wherein the cells are induced pluripotent stem cells.
 14. Amethod of promoting osteogenesis in a tissue, the method comprisingtreating the tissue with the composition of claim 1 in an amounteffective to promote the osteogenesis in the tissue, wherein the tissueis a bone tissue.
 15. The method of claim 14, wherein the osteogenicactivity of cells in the treated tissue is greater than the osteogenicactivity of cells in untreated tissue.
 16. A method of increasing acellular growth factor activity in cells, the method comprising treatingthe cells with the composition of claim 1 in an amount effective toincrease the cellular growth factor activity in the cells, wherein thecells are progenitor cells, adult stem cells, or induced pluripotentstem cells, and wherein the cellular growth factor activity isosteoinductive activity, chondroinductive activity, or a combinationthereof.
 17. The method according to claim 16, wherein the cells areprogenitor cells or adult stem cells.
 18. The method according to claim17, wherein the progenitor cells or the adult stem cells are derivedfrom placenta, bone marrow, adipose tissue, blood vessel, amnioticfluid, synovial fluid, synovial membrane, pericardium, periosteum, dura,peripheral blood, umbilical blood, menstrual blood, baby teeth, nucleuspulposus, brain, skin, hair follicle, intestinal crypt, neural tissue,or muscle.
 19. The method according to claim 16, wherein the cells areinduced pluripotent stem cells.
 20. The method according to claim 16,wherein the at least one protease comprises two or more proteases. 21.The method according to claim 20, wherein the two or more proteasescomprise: (a) a first protease selected from the group consisting ofcollagenase, clostripain, dispase, trypsin, MMP-13 (matrixmetalloproteinase-13), and a mixture thereof, and (b) a second proteaseselected from the group consisting of collagenase, clostripain, dispase,trypsin, MMP-13 (matrix metalloproteinase-13), and a mixture thereof,and wherein the first and second proteases are different.
 22. The methodaccording to claim 16, wherein the at least one protease is collagenase.23. A method of increasing a cellular growth factor activity in cells,the method comprising treating the cells with the composition of claim9, wherein the cells are progenitor cells, adult stem cells, or inducedpluripotent stem cells, and wherein the cellular growth factor activityis osteoinductive activity, chondroinductive activity, or a combinationthereof.
 24. A method of growing and/or culturing cells comprisinggrowing and/or culturing cells in vitro in the presence of thecomposition of claim
 1. 25. The method according to claim 24, whereinthe cells are selected from the group consisting of mesenchymal stemcells, adipose-derived stem cells, embryonic stem cells, progenitorcells, differentiated cells, undifferentiated cells, and inducedpluripotent stem cells.
 26. A method of making the composition of claim1, comprising preparing said peptide by contacting mature BMP-7 withcollagenase under conditions that promote protein cleavage to producethe peptide and harvesting the peptide, and mixing the peptide with theat least one pharmaceutically acceptable carrier and the at least oneprotease, whereby the composition of claim 1 is prepared.
 27. A methodof making the composition of claim 1, comprising preparing said peptideby culturing a host cell under conditions suitable for proteinexpression, wherein the host cell comprises a vector that encodes saidpeptide, harvesting the peptide, and mixing the peptide with the atleast one pharmaceutically acceptable carrier and the at least oneprotease, whereby the composition of claim 1 is prepared.